Modular organ microphysiological system with integrated pumping, leveling, and sensing

ABSTRACT

Fluidic multiwell bioreactors are provided as a microphysiological platform for in vitro investigation of multi-organ crosstalks for an extended period of time of at least weeks and months. The disclosed platform is featured with one or more improvements over existing bioreactors, including on-board pumping for pneumatically driven fluid flow, a redesigned spillway for self-leveling from source to sink, a non-contact built-in fluid level sensing device, precise control on fluid flow profile and partitioning, and facile reconfigurations such as daisy chaining and multilayer stacking. The platform supports the culture of multiple organs in a microphysiological, interacted systems, suitable for a wide range of biomedical applications including systemic toxicity studies and physiology-based pharmacokinetic and pharmacodynamic predictions. A process to fabricate the disclosed bioreactors is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalApplication No. 62/291,102 filed Feb. 4, 2016 and U.S. ProvisionalApplication No. 62/359,567 filed Jul. 7, 2016, which are herebyincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under ContractsW911NF-12-2-0039 and UH3TR000496 awarded by the Defense AdvancedResearch Projects Agency Microphysiological Systems Program and NationalInstitutes of Health, respectively. The government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

Improving the effectiveness of preclinical predictions of human drugresponses is critical to reducing costly failures in clinical trials.Complex diseases often arise from dysregulation of systemic regulatorynetworks, including across multiple organs, resulting from integrationof local and systemic perturbations. Incomplete understanding ofinter-tissue communication can undermine the accurate diagnosis andtreatment of disease conditions. Although the study of humanpathophysiology has relied on genetically tractable animal models suchas murine models, these animal models may be inadequate forrecapitulating polygenic and multifactorial human diseases with diverseclinical phenotypes.

Recent advances in cell biology, microfabrication and microfluidics haveenabled the development of micro engineered models of the functionalunits of human organs—known as organs-on-a-chip (OCC)—that could providethe basis for preclinical assays with greater predictive power. Forexample, U.S. Pat. No. 6,197,575 to Griffith, et al., describes amicromatrix and a perfusion assembly suitable for seeding, attachment,and culture of complex hierarchical tissue or organ structures. U.S.Pat. No. 8,318,479 to Inman, et al., describes a system that facilitatesperfusion at the length scale of a capillary bed suitable for cultureand assaying in a multiwell plate format.

These platforms, termed microphysiological systems (MPSs), are designedto mimic physiological functions by integrating tissue engineeringprinciples with microfabrication or micromachining techniques forrecapitulating 3D multicellular interactions and dynamic regulation ofnutrient transport and/or mechanical stimulation (Huh D, et al., LabChip, 12(12):2156-2164 (2012); Sung J H, et al. Lab Chip 13(7):1201-1212(2013); Wikswo J P, et al., Exp Biol Med (Maywood) 239(9):1061-1072(2014); Livingston C A, et al., Computational and StructuralBiotechnology Journal 14:207-210 (2016); Yu J, et al., Drug DiscoveryToday, 19(10):1587-1594 (2014); Zhu L, et al. Lab Chip, 16(20):3898-3908(2016)). While significant advances have been made in the development ofindividual MPS (e.g., cardiac, lung, liver, brain) (Roth A, et al., AdvDrug Deliver Rev, 69-70:179-189 (2014); Huebsch N, et al. ScientificReports, 6:24726 (2016); Domansky K, et al. Lab Chip 10(1):51-58(2010)), efforts towards the interconnection of MPS are still in theirinfancy, with most studies primarily focused on basic viability andtoxicity demonstrations (Oleaga C, et al. Sci Rep 6:20030 (2016); Esch MB, et al., Lab Chip 14(16):3081-3092 (2014); Maschmeyer I, et al., LabChip 15(12):2688-2699 (2015); Materne E M, et al. J Biotechnol 205:36-46(2015); Loskill P, et al., Plos One 10(10):e0139587 (2015)). However,lack of clinical efficacy, rather than toxicity, was identified as theleading cause of drug attrition in Phase II and III clinical trials (themost costly stage) (Kubinyi H, Nat Rev Drug Discov 2(8):665-668 (2003);Cook D, et al. Nat Rev Drug Discov 13(6):419-431 (2014); Denayer T, etal., New Horizons in Translational Medicine, 2(1):5-11 (2014)). Majorcontributing factors include incomplete understanding of diseasemechanisms, the lack of predictive biomarkers, and interspeciesdifferences. There is an urgent unmet need in drug development due tothe need for humanized model systems for targetidentification/validation and biomarker discovery.

The increasing need for more predictive in vitro systems is not limitedto single MPS technologies. The complexity of the human physiology canbe better recapitulated at a systemic level in multi-MPS platforms,where multi-organ crosstalk and the physiological responses totherapeutic agents and toxins occur via surrogate signals (e.g.chemokines, cytokines, growth factors) and circulating cells (e.g.immune cells). Shuler et al. demonstrated pharmacological applicationsof multi-compartmental bioreactor systems (Sweeney L M, et al., Toxicol.Vitr. 9, 307-316 (1995)). Sung et al. showed a micro cell culture analog(pCCA), where cells were embedded in 3D hydrogels in separate chambers,could be used for interacting MPS systems (Sung J H, et al., Lab Chip 9,1385 (2009)). Some prototypes use gravitational flow for inter-MPScommunication (Sung J H, et al., Lab Chip 10, 446-455 (2010)). Someprototypes of the three-MPS system use off-platform pumping with abubble trap (Sung J H, et al., Lab Chip 9, 1385 (2009); Esch M B, et al.Lab Chip 14, 3081 (2014)).

While toxicology and pharmacodynamic studies are common applications,pharmacokinetic studies have been limited in multi-MPS platforms.Moreover, current multi-MPS systems generally employ a closed formatassociated with traditional microfluidic chips for operating with verysmall fluid volumes (Anna S L, Annu. Rev. Fluid Mech. 48, 285-309(2016)). Current fabrication processes for these systems require the useof castable elastomeric polymers like PDMS mainly for desirable opticalproperties, but due to fluid-surface interactions such as drug andgrowth factor adsorption are commonly present (Halldorsson S, et al.,Biosens. Bioelectron. 63, 218-231 (2015)).

Other practical limitations in the design and fabrication of thehardware also significantly reduce the robustness, long-termreliability, and compatibility of customization in existing multi-MPSdevices. Poor hardware designs and constructs often result in a poor oflack of control on the directionality of fluid among wells (inter-welldirectionality) and within-well recirculation, leaving some wells drydue to breakage of fluid flow, the syphoning effect, and/or evaporation.Media depletion and waste removal at near-physiological scales oftenrequire single-pass media flow, making it difficult or impossible tostudy slow-clearing drugs, effects of drug metabolites, and inter-MPScommunications. Removable inserts to fit into the wells of multi-MPSdevices may be desirable in culturing some tissues, but theircompatibility with fluid in-flow to support perfusion of cultures hasbeen difficult to achieve.

It is therefore an object of the present invention to provide improvedapparatus with integrated fluid control means for long-term tissueculture and facile assaying of multiple modular organ models.

It is another object of the present invention to provide methods ofintegrating fluid pumping, leveling, and sensing with bioreactors.

It is yet another object of the present invention to provide insertdevices compatible with open fluid bioreactors, which support perfusionand allow off-platform seeding of cells and biomaterials, simplemanipulation, and easy removal from bioreactors without causing damageor contamination.

SUMMARY OF THE INVENTION

Multi-well cell culture systems (or organs-on-a-chip devices,microphysiosome bioreactors) are provided with integrated pumping,spontaneous liquid leveling, and programmable drug/media dosing. Amulti-well culture system, i.e., a chip or a bioreactor, contain atleast three layers of constructs, which from top to bottom are (1) amulti-well cell culture plate construct with built-in fluid channels(e.g., fluid paths) below and connected to the wells, (2) a barriermembrane as a pump actuator, and (3) a pneumatic plate to presentpressure and vacuum. In different embodiments, the membrane layer isbonded on either the fluidic or the pneumatic side, or is a separatecomponent. Bonding the membrane layer to the pneumatic or fluidic sideenhances reliability and reduces manufacture time and cost. In apreferred embodiment, the membrane is bonded to the pneumatic side, andthe fluidic layer is open faced, making cleaning and sterilizationeasier. In some embodiments, no bonding on the fluidic side eliminatesdelamination.

Pneumatic control of vacuum or pressure causes the membrane to actuate,which acts like a valve to control the passage or blockade on the fluidchannel, thus the fluid flow, on the fluidic side of the system. Fluidsuch as cell culture media is flowed in to fill at least one of thewells, and passive self-leveling spillways connecting two or more wellsin the upper space allow for transfer of excess fluid from one well toanother. Recirculation within a well or between two wells is allowedactively, through additional pumps.

The system combines one or more of the following features to improve theoperability and performance of modeled organs on a chip: Spillwayshaving defined geometric arrangements to promote unidirectional flow andanti-siphon capability. One or more features in the entry, the conduit,and/or the exit of the spillway are provided to ensure spontaneouscapillary flow across the spillway for unidirectional self-leveling offluid amount in MPS chambers. Some embodiments provide entry geometrythat eliminates a step or V-cut to minimize fluid film disruption; andincludes a radial meniscus pinning groove around the source well, thegroove being able to “pin” the fluid meniscus, making a specified fluidheight energetically favorable.

Some embodiments provide a spillway conduit that has a small-width(e.g., less than 3 mm), high aspect ratio groove at the bottom along theconduit to permit spontaneous capillary flow, thus leveling of excessfluid from the source well to the destination well. Some embodimentsprovide exit geometry where the groove at the end of the spillwayconduit encounters an enlarged, curved area, to thin the fluid film,thereby breaking it into drops which coalesce and fall due to gravity.In another embodiment, at the exit of spillway there is a verticalgroove along the wall and toward the bottom of the destination well.Some embodiments additionally provide an undercut into the wall of thedestination well, where the cut is at some distance below the exit ofthe conduit, to prevent back flow due to siphoning effect. Thesefeatures allow a self-leveling spillway in a unidirectional flow andprevent breakage of flow and over accumulation in the source well or theconduit.

Optionally coupled with an internal humidity reservoir or anevaporation-combatting moat, the multi-organ MPS platforms allow forlong-term culture of functional organ-like tissues, e.g., for at least1, 2, 3, 4, 5, 6 weeks or at least 1, 2, 3 months.

The on-board pumping system (e.g., built-in fluid pumping channels)eliminates the need for tubing, but modular pumping can be configured todrive external flows. Ferrule connections may be used to interface thebuilt-in pump with external tubing, allowing for a pumping manifold todrive a large number of flows simultaneously in a compact package.

A dual pumping system in addition to single multi-chamber unit pumpingsystem permits not only pulsating flow but also a smooth flow volumeprofile. A triple pumping system or more parallel channels may furtherincrease the smoothness of the flow.

A removable yet perfusion-enabled scaffold to fit into the wells on theplatform is provided. Unlike conventional removable inserts that do notallow integrable features to participate in the perfusion process in abioreactor, the scaffold enables cell culture to be perfused on-platformand processed off-platform. The scaffold may optionally contain a fluidaggregation lid for non-contact oxygen (O₂) sensing.

One or more means for non-contact fluid leveling sensing are provided.Capacitors with a symmetrical, front-and-back electrode design providesaccurate measurement of fluid level in a well from within the wall ofthe well, avoiding direct contact, electrochemical reactions, andpotential contamination.

Two or more multi-organ bioreactors may be daisy chained due to thepass-through design of internal channels (e.g., air actuation lines)passing through the body of the pneumatic plate of the bioreactor. Twoor more bioreactors may also be stacked to save space. Pneumatic lineand fluid connection layouts for stacked configuration are provided.

The platform is preferably fabricated from materials that minimize lossof biochemical factors due to adsorption. In some embodiments, the topfluidic plate is fabricated from polysulfone. In some embodiments, thetop fluidic plate is fabricated from polystyrene. In some embodiments,the pneumatic plate is fabricated from acrylic material. In someembodiments, the actuation membrane is fabricated from polyurethane;alternatively elastomers are placed on the multi-chamber pumping unit insections to replace the polyurethane membrane.

The organ-on-chip has on-board pneumatic microfluidic pumping in orderto achieve extended 3D culture of functional tissue such as livertissue. The on-board pumping technology minimizes space, auxiliaryequipment, and dead volumes associated with excess tubing. Thismulti-organ platform features deterministic pumping for precise flowrate control over a wide range of flow rates from 0 to several hundredsof milliliters per day with controlled volume flux such as between 0.1and 10 microliter per stroke, at frequencies between about 0.01 Hz and20 Hz, to provide controlled recirculation of medium within each MPS aswell as controlled “systemic” circulation.

The platform has a similar footprint to a typical multi-well plate withchambers designed to house different types of micro-tissues. Theindividual tissue compartments are equipped with their own intra-MPSpumps to provide nutrient recirculation and are fluidically connected tothe mixer via passive spillways for level control. Although one-organculture is feasible with the platform (e.g., with benefits of perfusionand drug addition coming from other wells), the hardware can bereconfigured to accommodate multiple applications including 2-way,3-way, 4-way and N-way interactions (N>=2), with user-defined control offlow rates and flow partitioning from the mixing chamber to thedifferent tissues, recapitulating physiologically-relevant circulation.

Validations of multi-way MPS interactomes are also provided. “M-W MPS”refers to a configuration whereby each individual micro physiologicalsystem has its own internal circulation to control oxygenation andmixing and mechanical stimulation independent of other MPS units on theplatform. Each MPS is connected fluidically to other MPS units in acontrolled manner via the central circulatory flow circuit, or viadirect connections. For example, the gut module has an internalcirculation to mix the fluid beneath the transwell membrane and receivesflow from the central circulatory flow, then its effluent goes directlyto the liver. The liver module has its own internal circulatory flow,and receives flow from the gut, the pancreas, and the centralcirculatory flow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary diagram of components in a multiwell device withon-board pumping system. A fluidic plate 100 contains two or more wells,which can be fitted with inserts such as a transwell 1101, fluid paths101 providing fluid connectivity between at least two of the wells, andpin holes or slots 102 for attachment with a second plate 200. Thesecond plate 200 (e.g., a pneumatic plate) contains a number of internalchannels (i.e., air actuation lines), each with openings 210 (an inletopening and an outlet opening) on opposing sides of the second plate200. On the surface of the second plate is one or more protrudingfeatures 201 corresponding to the shape, totuosity, and length of thefluid paths 101 of the fluidic plate 100. These protruding features haveholes in connection to each of the internal channel, such thatcompressed air or vacuum is distributed through the internal channels toholes on the surface of the pneumatic plate. The pneumatic plate alsohas slots 202 for attachment with the fluidic plate. Stainless steelscrews fasten the layers together into a single unit that can be handledlike a traditional N-well plate.

FIG. 2 is a schematic showing two devices daisy-chained at the openings210 of the internal channels (i.e., air actuation lines) of thepneumatic plate 200. The fluidic plate 100 (with a plate lid 1200) isassembled with the pneumatic plate 200.

FIG. 3A shows a schematic of an assembled 7-way device, having wells 103for cell culture and/or mixing medium where a transwell insert 1101 isfitted into a well. Two ports 105 in fluid connectivity with the fluidpaths of the fluidic plate may be used to connect with external fluidcontainers for import and/or export of fluid.

FIG. 3B is a map showing the organs to be placed and flow directionalitybetween organs on a 7-way platform corresponding to FIG. 3A.

FIG. 4 is a schematic of a top view of a pneumatic plate of a 7-waydevice. The plate has alignment pins 203 for alignment and slots 202 forattachment with a fluidic plate. The plate has protruding features 201on the surface which in multiple locations has a set of three holes,representing a set of three-chamber units 220 a, 220 b, and 220 c. Thesethree sets of three-chamber units are in air/pressure connection withthree internal channels (i.e., air actuation lines) with inlet andoutlet openings 210 a and 210 b on opposing sides of the pneumaticplate. The middle hole/chamber of each of these three sets ofthree-chamber units is positioned to share a same internal channel(i.e., air actuation line). The hole/chamber on the same (i.e., left- orright-hand side) of the middle hole/chamber of each of these three setsof three-chamber units is positioned to share another same internalchannel (i.e., air actuation line), reducing the complexity ofpneumatically actuated flow controls of the device. Correspondingpositions of a fluidic plate's wells and spillway conduit 121 are alsoshown on the pneumatic plate here.

FIG. 5 is a schematic showing a cross-sectional side view of agut-liver-lung-endometrium 4-way platform. Arrows represent thedirection of fluid flow, where fluid is pumped into a gut well 103 d viaan inlet 111 a in the well, and excess fluid above a height is spilledthrough a spillway conduit 121 to a liver well 103 b that contains anoxygenation tail 103 c. The gut well also has an outlet 111 b in thewell for potential same-well recirculation of fluid with inlet 111 a.Fluid from a mixer/mixing well 103 a flows through fluid paths to cellculture wells including an endometrium well 103 e and a lung well 103 f.The plate also has a moat 104 to combat evaporation.

FIG. 6 is a diagram showing the flow directionality and cell culturetype of each well on a 4-way platform operating in a two-wayconfiguration.

FIG. 7 is a diagram showing of the flow directionality and function ofeach well on a 4-way platform operating in a one-organ configuration.

FIG. 8 is a diagram showing the flow directionality, flow partitioning,and cell culture type of each well in a 2-way configuration.

FIG. 9 is a diagram showing the flow directionality, flow partitioning,and cell culture type of each well on another 4-way platform.

FIG. 10 is a diagram showing the flow directionality, flow partitioning,and cell culture type of each well on a 7-way platform.

FIG. 11 is a diagram of a different configurations of well orientationsfor drug additions to a 2-way interactome.

FIG. 12 is a schematic showing a top view of a spillway (containing aspillway conduit 121) providing unidirectional fluid connectivity from asource well 103 i to a sink well (or destination well) 103 j. The inlet111 a and outlet 111 b of the source well 103 i are also shown.

FIG. 13 is a side view of the entry geometry for a spillway from asource well 103 i (containing an outlet hole 111 b, e.g., for activepumping-induced recirculation). Radial meniscus pinning groove 122aligns with a curved entry geometry 124 of the spillway, and the curvedentry geometry aligns with the bottom of a conduit groove 125 of thespillway conduit. Transwell height is set by the vertical location of astep shelf 123 on which the outer rim of the transwell rests.

FIG. 14 is a side view of the exit geometry for a spillway 121 into adestination well 103 j. The exit geometry of the spillway includes anundercut 130 in the wall of the destination well, below the edge of thespillway conduit, and a vertical groove 131 to guide along the wall ofthe destination well.

FIG. 15 is a cross-sectional side view of a perfusable scaffold in aperfused well of a device showing the apical volume 1102 in the scaffoldand the basal volume 1103 in the well.

FIG. 16A, FIG. 16B, and FIG. 16C illustrate a successive time-course,potential development of a spillway V-shaped entry geometry of(cross-sectional side view), from initial continuous fluid film acrossthe spillway (FIG. 16A), to breakage of fluid film (FIG. 16B), andfinally drying in the sink well and over accumulation in the source(FIG. 16C).

FIG. 17 is a schematic of a cross-sectional side view of anotherspillway entry geometry without the V-shape in FIG. 16A, for continuousfluid film across the spillway.

FIG. 18 is a schematic of an enlarged cross-sectional side view of thespillway entry geometry corresponding to FIG. 17, i.e., U-shaped conduitwith a groove at the bottom.

FIG. 19 is a schematic of a cross-sectional side view of anotherembodiment of a spillway geometry. This spillway has a conduit 127 thatpermits open fluid flow (space above the conduit 126) with a towerconduit 128 a entry, and an upward conduit exit 129 a.

FIG. 20 is a schematic of a top view of the spillway shown in FIG. 19.The tower conduit has an opening, i.e., a hole 128 b, on the surface ofa step in the wall of the source well, which connects to the spillwayconduit 127 in an open-fluid configuration 126.

FIG. 21 FIG. is a schematic of a cross-sectional side view of thespillway shown in FIG. 19, where a screw 140 plugs the tower conduit 128a, preventing spillout flow from a source well.

FIG. 22A, FIG. 22B, and FIG. 22C illustrate a successive time-coursedevelopment of a spillway with a V-shaped entry geometry of(cross-sectional side view), from initial fluid front into the conduit(FIG. 22A), to migration of fluid front along the conduit (FIG. 22B),and fluid accumulation in conduit (FIG. 22C).

FIG. 23 is a schematic of the dimension of conduit geometry forcalculation to determine spontaneous capillary flow (SCF). W_(F) symbolsthe dimension of liquid-air interface.

FIG. 24 is a schematic of the dimension of a rectangle conduit forcalculation to determine SCF. The conduit has a depth of b and a widthof a, totaling a cross-sectional conduit perimeter of Pw, whereas theliquid-air interface has a perimeter of P_(F).

FIG. 25 is a schematic of a cross-sectional side of a spillway without aV-shaped entry geometry to support SCF.

FIGS. 26A-26D show different views of a rounded bottom spillway conduitat the inlet (FIG. 26A), a diagonal view (FIG. 26B), a section view(FIG. 26C), and at the outlet (FIG. 26D).

FIGS. 27A-27D show different views of a spillway conduit with a knifeedge geometry at the inlet (FIG. 27A), a diagonal view (FIG. 27B), asection view (FIG. 27C), and at the outlet (FIG. 27D).

FIGS. 28A-28D show different views of a spillway conduit with a V-cutgeometry at the inlet (FIG. 28A), a diagonal view (FIG. 28B), a sectionview (FIG. 28C), and at the outlet (FIG. 28D).

FIG. 29 is a schematic of the cross-sectional side view of a spillwayconduit geometry, i.e., U-shaped with a bottom-located rectangle grooveof a high depth-to-width ratio (e.g., greater than 3).

FIG. 30A, FIG. 30B, and FIG. 30C illustrate another successivetime-course development of a spillway with a V-shaped entry geometry(cross-sectional side view), from initial continuous fluid film acrossthe spillway (FIG. 30A), to fluid accumulation in the conduit (FIG.30B), and syphon effect (FIG. 30C).

FIG. 31 is a schematic of a cross-sectional side view of a spillway exitgeometry, where the spillway conduit 127 ends with a slope 132, and adistance of d below the conduit there is an undercut 130 in the wall ofthe destination well. A vertical groove 131 below the slope 132 andinterrupted by the undercut 130 is present along the wall of thedestination well.

FIG. 32 is a schematic of a top view of a spillway exit geometry wherefluid flowing from a small-width groove 127 encounters an enlargedcurved area 132 for exit.

FIG. 33 is a schematic of a top view of an oxygenation tail 150 a withguiding grooves 151 on the bottom surface of the well.

FIG. 34 is a schematic of a top view of a well 103 connecting to azig-zag oxygenation tail 150 b.

FIG. 35 is a diagram showing the geometry features of the zig-zagoxygenation tail shown in FIG. 34, for a phase-guiding purpose. The tailhas a maximum width of W₁ and a minimum width of W₂, appearing in analternating order for a length of L₁ and L₂, respectively. The angle αsymbols the direction of an increasing width with respect to the fluidflow direction in the oxygenation tail.

FIG. 36 is a schematic of the cross-sectional side view of a removable,perfused scaffold 160 inserted into a well on platform, which shows aramp area 159 for securing (e.g., turn by screw thread) the scaffold,radial seals 161 a and 161 b (e.g., O-rings), a cell culture region 162in the scaffold, and a fluid aggregation lid 163 useful for non-contactoxygen sensing.

FIG. 37A is a schematic showing the top view of a three-chamber unit onthe surface of a pneumatic plate.

FIG. 37B is a schematic showing the side view of a three-chamber unitcorresponding to FIG. 37A. A barrier membrane 300 separates a fluidicplate (containing a fluid path 101) and a pneumatic plate. The pneumaticplate has protruding features 201 on which holes create chamber spacesthat are connected to internal channels (air actuation lines) of thepneumatic plate (not shown in this Figure). Here the chamber 221 servesas a valve, chamber 222 as a pump, and chamber 223 as another valve.

FIG. 38 is a schematic of a top view of split fluid flow on top of dualthree-chamber units that are controlled by four air actuation lines.

FIG. 39 is a cross-sectional side view schematic of an in-wall fluidlevel sensing capacitor 1200, including front electrodes 1201 and backelectrodes 1203 that are on opposing sides of a board 1202 (e.g.,polychlorinated biphenyl (PCB) board).

FIG. 40 is a top view schematic of the electrodes of the in-wall fluidlevel capacitive sensor shown in FIG. 39, showing a front sensingelectrode 1201 b with a front reference electrode 1201 a coplanar on oneside and another front reference electrode 1201 c coplanar on the otherside, as well as a back sensing electrode 1203 b with a back referenceelectrode 1203 a coplanar on one side and another back referenceelectrode 1203 c coplanar on the other side.

FIG. 41 is a schematic of three layers of pneumatic lines for stackedplatform.

FIG. 42 is a cross-sectional side view schematic of a top plate 150 anda bottom plate 250, with geometries supporting sintering between the twoplates. The bottom plate 250 has protruding pillars 251 a and 251 b withnarrowed vertices 252 a and 252 b, respectively, and flat surfacedprotrusion 253 lower than the protruding pillars by a height of d₂.

FIG. 43 is a cross-sectional side view schematic of a fused, one-piececonstruct 350, sintered from the top plate 150 and bottom plate 250 ofFIG. 42. The vertices of protruding pillars in FIG. 42, after sintering(forced compression between the top plate and the bottom plate underheat), have deformed into sintered surfaces 252 c and 252 d and attachedwith the top plate. Space between protrusions of a bottom plate beforesintering has become space (e.g., channel) for fluid 351.

DETAILED DESCRIPTION OF THE INVENTION I. Definition

The terms “organ-on-chip (OOC)”, “bioreactor”, and “microphysiologicalsystem (MPS)”, used interchangeably, refer to the platform providing forinteractions among single or multiple organ or other tissue types on anin vitro platform which provides for the maintenance of growth of thesetissues.

The term “pneumatic” refers to a system which uses air or vacuumpressure for operation.

The term “manifold” refers to an interconnection device for pneumatic orfluid connections.

The term “spillway” refers to a system of fluidic connections between asource well and a destination well to automatically maintain fluidlevels in the source well.

The term “leveling” refers to maintaining fluid level.

The term “self-leveling”, refers to maintaining level using passivemeans, i.e., without active means.

The term “undercut” refers to a mechanical detail associated with anoverhanging feature.

The term “wetting” refers to the wetting of a solid surface by a liquidin a gas environment, which is determined by the minimum in Gibbs energyof the system. Wetting of a solid surface by a liquid in a gasenvironment results in an equilibrium contact angle θ across the liquidphase between the solid/liquid (SL) and liquid/gas (LG) interfaces asthey emanate from the contact line. Generally the terms “wetting” and“nonwetting” surface refer to cases of θ<90° and θ>90°, respectively.The relationship between the contact angle and the interfacial energiesinvolved is expressed by Young's equation γ_(SV)=γ_(SL)−γ cos θ, whereγ_(SV), γ_(SL), and γ are the Gibbs interfacial energies between solidand gas, solid and liquid, and liquid and vapor, respectively, and wherethe last quantity is addressed as surface tension. To satisfy thethermodynamic equilibrium requirement, the gas phase is saturated withvapor.

The term “meniscus” refers to the fluid boundary at the intersection offluid with a solid material and a vapor phase.

The term “meniscus pinning” herein refers to, in a situation of raisingthe level of a wetting liquid in a vertical well to the top edge, theend of the wetting line with a contact angle θ stays (or “is pinned”) atthe top edge of the well while the contact angle θ to rise from <90°to >90° at the top edge of the well side wall during further increase ofthe liquid level, until accumulation of liquid results in spilling overthe edge of the well, thus releasing the contact line (“unpinned”). Fornonwetting liquid, meniscus pinning occurs at the base edge and the topedge of the side face of a vertical well, and at the top edge the anglefor the liquid orientation at the contact line changes from the value θto the value θ+90°. Details of the term is described in Wijs et al.,Separations: Materials, Devices and Processes, 62(12):4453-4465 (2016).

The term “capillary length” refers to a characteristic length scale foran interface between two fluids which is subject both to gravitationalacceleration and to a surface force due to surface tension at theinterface.

The term “insert” refers to an element which can be mechanicallyassembled in a well of an MPS.

The term “scaffold” in the relevant sections is an insert or componentof the wells which provides support for tissue constructs.

The term “whippletree” refers to a mechanism to distribute force orpressure evenly through linkages. As used herein, it refers to force orpressure applied from one direction at or near the center anddistributes to the tips (generally two tips), where each serves as thecenter for distribution to further tips.

The terms “program” or “software” refer to any type of computer code orset of computer-executable instructions that can be employed to programa computer or other processor to implement various aspects ofembodiments as discussed above. Additionally, it should be appreciatedthat one or more computer programs that when executed perform methods ofthe present invention need not reside on a single computer or processor,but may be distributed in a modular fashion amongst a number ofdifferent computers or processors to implement various aspects of thepresent invention.

II. Apparatus and Operation of Apparatus

Each multiwell device is generally a three-component construct with anon-board pumping system. A fluidic plate 100 contains multiple wells,some be fitted with inserts such as a TRANSWELL® 1101 (Corning,distributed also by Sigma-Aldrich), and built-in micromachined fluidpaths 101 for distribution of culture medium (FIG. 1). A pneumatic plate200 distributes compressed air and vacuum to the surface of thepneumatic plate through small holes. A barrier membrane 300 (generallytranslucent) is situated between the fluidic plate 100 and the pneumaticplate 200, which under pressure may flex to expand or contract, therebyobstructing or clearing corresponding portions of the fluid paths of thefluidic plate. This barrier membrane also provides a sterile barrier,acting as the actuation layer of the pumps and valves.

Multiple devices can be chained for simultaneous in-phaseoperation/actuation (FIG. 2). Each device is a bioreactor, which as aplatform supports the culture of multiple MPSs mimicking differentorgans, their interconnections, and interactions as in vivo. The openwells and channels allow users easy access to the cells and culturemedia to perform measurements requiring direct fluid contact. Up toseven of these MPS have been coupled together, as demonstrated in theexamples, although it is understood that the system allows for mixing ofmore than one of the same type of MPS as well as mixing and integrationof a variety of different types, not limited to a total of seven.

The system uniquely incorporates a high degree-of-freedom (DOF) on-boardpumping system, effectively configured to support multiple organculture. While existing devices have compartments linked linearly by asingle pump to drive flow through a loop (Materne E M, et al., J. Vis.Exp. 1-11 (2015). doi:10.3791/52526) or linked in parallel with channeldiameters imposing predefined passive flow rates (Oleaga C., et al.,Sci. Rep. 6, 20030 (2016)), a high DOF control makes it easy toreconfigure the platform for addition of new MPSs or exclusion ofcertain compartments.

In some embodiments of 4-way MPS bioreactors, the platform may operatewith 18 degrees of freedom (“DOF”), or 18 individual channels of tubing.For example, in a liver-gut-lung-endometrium 4-way MPS, an individuallyaddressable pump requires 3 DOF, while multiple pumps can be run at thesame rate by sharing inlets on the pneumatic manifold across multiplepumps. A 4-way MPS platform may have 6 independently programmable flowrates which are used to drive 9 pumps. All four pumps providingmixer-to-MPS flow can be individually addressable. Recirculation pumprates are shared: mixer/liver recirculation are linked, as aregut/lung/endometrium recirculation. It is economically advantageous tolink pump rates, as this reduces the number of pneumatic valves andtubing connections required for a platform.

In some embodiments of 7-way MPS bioreactors, the platform has 36 DOFswhich operate the functional equivalent of 17 syringe pumps perplatform, and can dynamically control intra- and inter-MPS mixing. Inthis instance, only 12 flow rates can be independently specified, aseach requires 3 pneumatic lines.

A. Multi-well Bioreactor (1) Overview of Directions of Fluid Flow

FIG. 3A shows a schematic of a 7-organ interactive bioreactor, for whichFIG. 3B shows an exemplary map of tissues to be cultured in each welland directions of fluid flow. In an exemplary 7-way bioreactorcontaining lung, endometrium, gut, liver, heart, central nervous system(CNS), and pancreas, generally active flow of fluid is conducted viabuilt-in fluid channels from the mixer well (Mixer) to lung (arrow 1 inFIG. 3B), from Mixer to endometrium (Endo; arrow 3 in FIG. 3B), fromMixer to gut (arrow 4 in FIG. 3B), from Mixer to liver (arrow 7 in FIG.3B), from Mixer to pancreas (arrow 9 in FIG. 3B), from Mixer to CNS(arrow 10 in FIG. 3B), from Mixer to heart (arrow 11 in FIG. 3B); andvia within-well pumping to recirculate within each of lung, endometrium,gut, heart, CNS, liver, pancreas, and Mixer (arrows 2, 6, and 12 in FIG.3B). External supply may be imported to Mixer (arrow 8 in FIG. 3B),which through the fluid flow gets distributed to each organ well. Wastefrom Mixer may be exported to an external collector (arrow 5 in FIG.3B). In some embodiments, each out-flow from Mixer to an organ has adesignated pump for individually controlled flow rates, as well as theexternal supply import to Mixer and the export of waste to externalcollector from Mixer. To reduce complexity in some embodiments, therecirculation within each of lung, endometrium, and gut may share onepump control for an identical recirculation flow rate; the recirculationwithin each of heart, CNS, and pancreas may share another pump controlfor an identical recirculation flow rate; and the recirculation withinMixer and within liver may share yet another pump control for anidentical recirculation flow rate.

Spillways are generally designed between at least one pair of wells, andin one embodiment of the 7-organ platform between lung and Mixer,between endometrium and Mixer, between gut and liver, between liver andMixer, between heart and Mixer, between CNS and Mixer, and betweenpancreas and liver, to automatically transfer excess fluid from theformer well to the latter.

FIG. 4 shows a schematic of the pneumatic bottom plate corresponding tothe exemplary 7-way apparatus shown in FIG. 3A for multi-organ cultureas mapped out in FIG. 3B. A pneumatic plate may have alignment pins 203,in some embodiments two pins at symmetrical positions about the center,on the side of the pneumatic plate for mating/aligning withcorresponding features (e.g., pin holes or slots) on the bottom of thetop plate. A pneumatic plate may also have a number of holes 202throughout the depth of the plate, on multiple locations (notobstructing the air-conducting actuation lines), for correspondingprotruding pin features on the bottom of the top fluidic plate to alignwith. On the pneumatic plate shown in FIG. 4, there are 18 internalchannels as air-conducting actuation lines spanning horizontally acrossthe inside of the pneumatic plate. For example, a set of threeair-conducting actuation lines with air inlets and air outlets 210 a and210 b (entry and exit being relative to the orientation of the plate)controls multiple three-chamber units 220 a, 220 b, and 220 c that arelocated on the surface of the actuation-side (i.e., the side thatthrough an actuation membrane assembles with the bottom of the fluidicplate) of the pneumatic plate. Each three-chamber unit (e.g., bracketedas 220 a, 220 b, and 220 c) has three chambers, each having anair-conducting hole to the surface connecting with a horizontalair-conducting line below, and three chambers as a whole controls, viapneumatic actuation causing plus and minus deflection of a membrane, thestroke or the peristaltic fluid flow in the fluid channel of a top plateonce assembled. The pneumatic plate may also have protruding curved lineraised features 201 connecting one or more three-chamber units. Theseraised features provide the matching sealing surface for thecorresponding fluidic channels in the bottom surface of the fluidicplate which conduct fluid in defined fluidic circuits interconnectingthe various fluidic MPS modules. These raised features 201 can be seenoutlining the positions of fluidic paths in a fluidic plate once thepneumatic plate is assembled with a fluidic plate. Element 121 shows theposition of the spillways which carry fluid between the MPS modules in afluidic plate, once the pneumatic plate is assembled with a fluidicplate.

FIG. 5 shows a cross-section of an exemplary 4-way platform showing abuilt-in channel for fluid flow from mixer to gut, and a generalspillway position from gut to liver. The disclosed wells for cellculture on the multi-organ MPS platform generally follow this“flow-in/spill-out” principle of operation.

Operation of the directions of active flow and passive spillover offluid generally mimic circulation paths in in vivo systems, and theprinciples as shown in the exemplary 7-organ bioreactor are applicableto platforms of 2-way, 3-way, 4-way, or other numbers of MPS systems.Exclusion of one or more wells from use in a multi-well platform isfeasible via alteration in software code for operation, and no hardwarechange is required. Each well is also reconfigurable for multiple uses.For example, a mixing chamber (Mixer well) may also be used asimmune-competent gut MPS well, or be used with a TRANSWELL®. A liver MPSwell may be used as a media reservoir or drug reservoir. Exemplaryreconfigured use of a multi-well platform is shown in FIGS. 6-10. Flowpartitioning is generally achieved by varying the frequency of pumping.Another exemplary configuration of multi-well platform is shown in FIG.11, where three drugs housed in three wells are delivered to liver welland gut well, while the wells are perfused and in interaction via Mixerwell and the spillway between liver and gut.

(2) Means for Controlling Flow Direction and Level Self-LevelingSpillways

The apparatus achieves self-leveling of MPS wells passively and fluidreturn, generally to Mixer, by a system of spillway channels cut intothe top side of the plate to deliver excess fluid back to the mixer. Ingeneral, a spillway includes a channel (e.g., open fluid) above certainof the bottom wells, which connects an inlet well to an exit well (FIG.12). Spillways eliminate the need for return pumps and level sensors forenforcing a balance between influx and efflux, while also allowingreturn flows to cross over the inlet MPS feed flows. In preferredembodiments, the spillways avoid breakage of fluid flow in the spillwaywhen leveling is needed, and avoid the siphon effect to prevent dryingout of wells.

The apparatus uses spontaneous capillary flow (self-wetting) and phaseguiding principles to guide flow and wetting in fluid pathways to allowfor more robust operation of open fluidic organ-on-chip systems.Unidirectional flow from a source well to a destination well is achievedwith meniscus control features, detailed below, and othercharacteristics including additional groove geometry of the spillwayconduit, controlled surface roughness, surface tension, and additionalfeatures in the entry and exit of the spillway. These one or moregeometric features in fluid containers for the organs-on-chips apparatusallow for pinning of fluid in a radial fashion to limit the meniscuseffect created by surface tension. This construction could allow forbetter passive fluid leveling which could then translate in moredeterministic performance and measurement within these systems.

The spillways implement passive leveling in the following fashion. Iffluid flow into the inlet well causes a net accumulation of fluid in theinlet well, the level in the inlet well will begin to rise. As the levelbegins to rise, the fluid will rise at the spillway, and thereby causeincreased flow through the spillway into the exit well. If the level inthe inlet well decreases, the fluid level at the spillway of the inletwell will drop, thereby decreasing the flow through the spillway. Inthis manner, the level in the inlet well is passively controlled to beapproximately equal to a desired level. Such leveling is passive in thatthere is not an active process of sensing level and changing somepumping rate in response to this sensing of level. Rather the effects ofgravity and surface tension combine to regulate flow in a passive mannernot requiring explicit sensing and control.

To achieve proper spilling function, the spillway employs a lowresistance flow path in the direction from source to sink, above thedesigned height of fluid in the source. In some embodiments, the path isimpermeable to flow in from the sink to the source and the system, suchthat as a whole the spillway may be resistant to transient changes influid height due to tilting.

Entry Geometry

Various inlet features are useful for stabilizing the source wellmeniscus, providing an entry into the spillway channel or a way ofsealing the volume of the media in the source well.

FIGS. 16A-16C show a time-course schematic of how a spillway with aV-cut at the source well (inlet well) experiences discontinuation of thefluid film (e.g., fluid film breaks) and thus the spillway conduitdries, causing fluid to accumulate in the source well and the sink wellto dry until empty. This type of spillways start off operating in ametastable regime with a connected fluid profile that allows fluidtransport. When fluid film breaks (specifically at entry step and V-cutgeometries, the fluid finds it more energetically favorable toaccumulate in the source well, thus increasing in height, rather than toadvance in the spillway entry and spillover into the conduit and sinkwell (outlet well). When the height increases beyond a certain value, iteventually spills over; but for organs having large surface area, suchas pancreas and liver, this increase in height requires a large amountof volume, which was found to be a major reason for the mixer to dry outafter 12 hours in incubator in testing of the 7-way platforms usingthese geometries.

The following have been determined to improve efficacy:

Shallow and Gentle Entry for Flat Meniscus

Shallower and gentler entry geometry to the spillway minimizes energyfor spilling fluid into conduit groove. A radial groove in the sourcewell directs meniscus and makes use of height increases to producespilling events. When fluid film is present and spillways are conductingfluid, the step and V-cut features may not prevent volume displacementfrom transient tilting or siphoning. Therefore, for some embodiments, anentry step and a V-cut are eliminated to minimize fluid film disruptionat this level. Step barriers may be used to prevent further fluidbuild-ups, as shown in FIG. 17 with a cross-sectional view of anexemplary entry without the V-cut shown in FIG. 18.

When gravity dominates and surface tension effects are negligible as inlarge wells with larger interconnecting spillways, V-cuts are effectivein determining the exact height of self-levelling and breaking theconnection. For smaller geometries, it is more effective to have adirect entry into the spillways (and in one embodiment, have a meniscuspinning groove) and take care of breaking the fluid contact by the useof spillway exit features.

Fluid-Pinning Groove

In some embodiments, the entry to the spillway additionally includes a“fluid pinning” groove, which can be a 20-, 30-, 40-, 45, 50-, or60-degree circumferential groove 122, preferably 45-degree, in the fluidwells. This groove captures the fluid meniscus, which facilitatesmaintaining a defined fluid height and improves the dynamics of levelingand spillway operation. The bottom of this radial meniscus pinninggroove aligns with the bottom of the spillway fluid flow channel asdetailed in FIG. 13. The pinned meniscus is unstable, and thus willspill over, so that the fluid does not rise beyond the height of theradial meniscus pinning groove.

Insertion of Teflon Rings for Deterministic Fluid Level.

Placing Teflon rings at different heights relative to the spillwaydetermines the maximum fluid height before spilling. An inserted Teflonring captures meniscus, therefore securing the liquid level not to gopass it. The ring also helps prevent evaporation.

Embodiments

FIG. 13 shows one embodiment of the improved entry geometry for thespillway, in which a shallow and gentle entry of fluid via a radialmeniscus pinning groove around the well, where the bottom of themeniscus pinning groove aligns with the bottom of a grooved fluid flowchannel.

FIGS. 19 and 20 show another embodiment of an improved entry geometryfor an open conduit spillway in a cross-sectional side view and a topview, respectively. A slanted conduit tower 128 a connects the sourcewell to an open conduit 127, which may have a spontaneous capillary flow(SCF) groove at the bottom. The entry geometry utilizes ahole-in-the-wall design, where a hole 128 b is created on a step surfaceto connect to the slanted conduit tower 128 a. A screw seal 140 may beplaced to plug the opening hole of the conduit 128 a to isolate MPSinteractions (FIG. 21). The screw seal generally has an O-ring next tothe thread to create a good seal once plugged into the hole.

Conduit Allowing for Spontaneous Capillary Flow (SCF)

FIGS. 22A-22C illustrate a time-course development of fluid across thespillway conduit from a spillway with a V-shaped entry geometry. Whenthe conduit has not been primed or when spillway conduit is dry due toevaporation or fluid film disruption, the front of a migrating fluidcoming from the source well forms a meniscus within the wall of theconduit, which advances slowly and accumulates fluid above the groove ofthe conduit. This spillway conduit issue was first observed in dyetesting on a 7-way alpha spillway, where the spillway was wetted byfluid front but the fluid migration along the conduit was slow andrequired substantial volume to wet the entire spillway.

The following represent means for improving flow by altering conduitgeometry.

Geometry and Dimension to Allow Spontaneous Capillary Flow to AssureRobust Wetting in Channels.

The fluid movement efficiency along the channel was compared among around-bottom, a V-shaped, and a rectangle-bottom open channel of acomparable small dimension. 2 μL of fluid droplet was added at one endof the open channel to measure the wetting distance without priming ofthe channel. A V-shaped channel was shown to exhibit a wetting distanceof 103 mm; a rectangular shaped channel had a wetting distance of 44 mm,and a round-bottomed channel had a wetting distance of 7 mm. Both theV-shaped channel and the rectangle-bottom channel support Concus-Finnflow (Berthier J, et al., AIMS Biophysics, 1(1):31-48 (2014)). A greaterwetting distance generally shows a greater wettability performance whichmaintains a continuous fluid flow in an open channel spillway.

Effect of Material Used to Form the Conduit

A conduit with spontaneous capillary flow (SCF) maintains a fluid filmand thus fluidic communication with minimal volume requirements andwithout any particular priming or pumping rate. To achieve SCF, thecross-section of the conduit should satisfy the following relationship:

${\frac{p_{F}}{p_{W}} < {\cos \mspace{11mu} \theta}},$

where

P_(F)=The free (in contact with air) perimeter

P_(W)=The wetted (in contact with wall) perimeter

θ=The generalized Cassie angle (the average contact angle of thematerial).

SCF results when the energy reduction from wetting walls outweighs theenergy increase from extending the free surface. Using Gibbsthermodynamic equation, the general criterion for spontaneous capillaryflow in composite-wall and air systems is the generalized Cassie angle θmust be <90°. The generalized Cassie angle is the average contact angleof the material. In preferred embodiments where the fluidic plate ismade with polysulphone, the contact angle for media-polysulphone-air hasbeen measured to be 30°<θ_(c)<113° for polysulfone with water or media.This wide range of contact angles is based on the effects of surfacemicro pattering and in lesser degree small differences in polysulfonehydrophobicity and thermal effects of incubation environments. Tosatisfy the SCF relationship, the range of perimeter ratios that allowfor SCF in the embodiments described herein ranges from0<P_(F)/P_(W)<0.866 (cos 30°≈0.886; negative perimeter ratios are notpossible, thus not considered). This is an exemplary estimation, and itis to be understood that other implementations may utilize alternativeratios. Practically, the contact angle anywhere in a channel isreasonably assumed to be ≦80°, considering the meniscus effect and/orpoorly wettable surface (which may be machined to generate a smoothfinish to encourage higher wettability). Therefore in a scenario with aprominent meniscus effect, or with poorly wettable surfaces, such thatthe contact angle is about 80°, the perimeter ratio goes0<P_(F)/P_(W)<0.18 (cos 80°≈0.174; arccosine 0.1866≈80°) in order tosatisfy the SCF relationship.

FIG. 23 and FIG. 24 provide a cross-section analysis of a channel of anarbitrary shape. Here, the perimeter of liquid exposed to air, W_(F),would be the free perimeter, P_(F), in the above relationship; and thesum of liquid perimeter in contact with three walls, W₁+W₂+W₃, would bethe wetted perimeter, P_(W), of the above relationship. FIG. 24illustrates an exemplary rectangle shaped channel with a width of a anda height of b. To satisfy the SCF relationship, the perimeter ratioshould follow:

${\frac{p_{F}}{p_{W}} = {\frac{1.5a}{\left( {{2b} + a} \right)} < {\cos \mspace{11mu} {\left( {80{^\circ}} \right).}}}}\mspace{11mu}$

When defining an aspect ratio, λ=b/a, therefore b=λa, the relationshipgoes

${\frac{1.5a}{\left( {{2a\; \lambda} + a} \right)} < 0.18},$

which can be calculated to derive a criterion for the aspect ratio toallow SCF by a poorly wettable surface and/or a channel surface with aprominent meniscus effect:

$\lambda = {\frac{b}{a} > 3.7 \approx 3.}$

Therefore, a small rectangle channel with an aspect ratio greater than 3generally can achieve SCF.

In some embodiments considering manufacturing capabilities, the aspectratios range is 2.5<λ<5 to support the SCF design principle.

In some embodiments, spontaneous capillary flow is achieved in atriangular horizontal channel with an aspect ratio of about 2, where thewall smoothness is such that the contact angle is about 60°. Thecalculation of P_(F)/P_(w) for a triangular channel would be differentcompared to a rectangular channel, but the same principles hold.

In some embodiments, a preferred fluid path within the spillway conduitis a rectangle or V-shaped channel with an aspect ratio greater than 3,which is within microfluidic dimensions to allow for capillary flow tooccur (FIG. 25 showing a continuous fluid film across the spillway).Upon an initial fluid contact with the conduit channel, a minimal volumeof fluid in a channel with a geometry supporting SCF will quickly wetthe entire geometry and produce a fluid film capable of efficientlytransporting fluid from source to sink.

Capillary Length and Spillway Width to Assure Gravity DependentSpilling.

According to Brakke et al., Exp Math, 1(2):141-165 (1992), for water incontact with acrylic (which has a similar hydrophobicity topolysulphone), the capillary length, [γ/(ρ*g)]^(1/2) (where γ is thesurface tension, ρ is the density of the liquid, and g is gravityacceleration), is 2.7 mm. If the distance between the two walls of achannel (i.e., width of the spillway channel) is less than the capillarylength, gravity has a negligible effect. Therefore, a spillway width of2.1 mm places the system in a regime where gravity is less dominant thancapillarity.

In some embodiments where spilling is desired to be driven by gravity(e.g., in conduit tower 128 a), the spillway width is greater than 3 mm.

Embodiments

FIG. 29 shows an exemplary spillway conduit geometry with a 3:1 aspectratio rectangle-shaped groove to allow for spontaneous capillary flow.U-shaped channel above spillway is a relief cut to allow space for thedrill bit collet.

In preferred embodiments, the surface tension spontaneously propagatesonce the liquid in the source well is leveled, and drives movement offluid through the conduit to the target well.

Exit Geometry with Undercut Design

FIGS. 30A-30C illustrate spillway exit from a spillway with a V-shapedentry geometry and no additional exit geometry. When the spillway exitdoes not have a fluid film in the vertical wall, fluid startsaccumulating in the conduit and leads to spilling bursts or even astable meniscus at the exit geometry. This accumulation stops when themeniscus of fluid at the conduit makes contact with the meniscus at thesink, and a fluid film is reestablished. When fluid film is alwayspresent, a poor exit design may see the siphon effect even after thesource fluid level is below the sink level.

This problem can be avoided or minimized using one or more of thefollowing options:

Sharp Undercut Along a High Aspect Ratio Vertical Groove to PreventBackward Flow.

FIG. 31 illustrates the spillway conduit 127 exits, via a slightlytapered, shallow slope (edge) 132, to connect with a vertical groove 131along the wall of the sink/destination well. A sharp undercut 130, e.g.,made with a milling machine, breaks the vertical groove 131 into twoparts. The undercut is a cut into the wall of the sink well below thetapered, shallow slope 132, and has an angle from the vertical line ofgreater than about 30° (e.g., 30°, 35°, 40°, 45°, 50°, 55°, 60°, ormore, and any continuous angle in between the exemplary numbers). Insome embodiments, the distance between the undercut 130 and the spillwayconduit exit, d, is between about 5 to about 10 times the width of thespillway conduit, in order to prevent the syphon effect. The verticalgroove 131 is designed to exhibit spontaneous capillary flow (SCF) andto maintain a fluid film. The vertical groove runs continuously from topto bottom, except where the undercut is present. This geometry helpsmaintain a stable fluid film connecting the conduit and sink as long asthere is forward fluid directionality. In case of reverse flow (e.g.,the syphon effect), the undercut cuts the fluid film and generates afluid meniscus that will only re-connect the fluid film when forwardflow is reestablished.

In some embodiments, the spillway exit vertical groove is configured toexhibit spontaneous capillary flow (SCP) using the same designparameters described in the SCF groove in the conduit, e.g., a highaspect ratio greater than 3. The undercut and the high aspect ratiovertical groove have been tested in a series of experiments in 3×3 alphaspillways and machined polysulfone block, leading to a controlled fluidfilm breakage and anti-syphoning effect. A stable vertical fluid film onthe improved exit geometry does not easily evaporate and allows forfluid film restoration and flow upon forward flow at spillway exit isresumed.

Rounded Slope Exit and Small-Width Groove to Break Film into Droplets.

Another improved feature is to introduce a rounded slope exit/edge atthe end of the spillway conduit. When the small-width SCF groove of thespillway conduit “meets” an enlarged, round-curved area (FIG. 32), thestable liquid film in the small-width SCF groove (due to surfacetension) becomes unstable at the enlarged round curved exit area, whichis effectively broken into droplets and would fall (“sheds”) into thesink well. This way, the source well becomes independent from the sinkwell, and unidirectionality of fluid flow is achieved.

In some embodiments, the entry geometry to the conduit from the sourcewell has no slope, i.e., it drops from a sharp edge, while the exitgeometry from the conduit encounters an enlarged, curved area, beforeliquid drops into the sink well.

Alternative Upward Exit from the Conduit

In some embodiments where the SCF channel is below the desired liquidlevel in the sink well, an upward exit conduit with an exit hole isutilized, as shown by element 129 a of FIG. 19.

Embodiments

FIG. 31 illustrates the spillway exit with a undercut beneath the exit,and vertical groove for anti-siphon effect.

Wall-bound drops that are pinned on an edge of a planar wall aregenerally referred to as wall-edge bound drops. Wall-Edge bound dropsare typically found in nature as dew hanging from the leaves of plantsuntil a sizable volume is reached and the drop falls. When drops arepinned on a pointed wall edge, they are referred to aswall-edge-vertex-bound drops. Wall-edge-vertex-bound drop simulationsshow liquid interfaces in contact with highly wetting solid walls(forming a spillway exit) tend to drip as the angle decreases. This isbecause the energy decrease from wetting the walls is greater than theenergy of the liquid-air interface, such that the contact area wants toexpand indefinitely in corners with smaller angles where thin fluidfilaments form. The creation of a thin fluid filament is relevant anddesirable in situations where accurate control of fluid leveling andflow is needed for open-channel fluidic systems, as the meta-stabilityof these filaments can provide means to allow or stop fluid transport.

(3) Recirculation

Passive self-leveling may contribute to return of flow as described indetail above.

Typically, recirculation is used to ensure that within a well, theconcentrations are well distributed and uniform. Thus, recirculationflow-rates are typically higher than organ to organ flowrates.

Active recirculation, driven by within-well pumping, may increaseoxygenation of the media. For example, recirculation may take placewithin each of lung, endometrium, gut, heart, CNS, liver, pancreas, andMixer in a 7-way MPS platform. To reduce complexity in some embodiments,the recirculation within each of lung, endometrium, and gut may shareone pump control for an identical recirculation flow rate; therecirculation within each of heart, CNS, and pancreas may share anotherpump control for an identical recirculation flow rate; and therecirculation within Mixer and within liver may share yet another pumpcontrol for an identical recirculation flow rate.

(4) Features to Encourage Oxygenation

Adequate perfusion rates to “meso-scale” tissues, commonly containinghundreds of thousands to many millions of cells, is difficult andcritical to cell viability. Based on the oxygen consumption rate ofliver, which has a high oxygen requirement, using cell culture medium asthe circulating fluid, a flow rate between about 6 and 10 μL per secondis needed per million of cells (Powers M J, et al., Biotechnol Bioeng78, 257-69 (2002); Domansky K, et al., Lab on a Chip 10, 51-58 (2010);Ebrahimkhani M R, et al., Advanced Drug Delivery Reviews April, 132-57(2014)). Because gas exchange can occur at the air-liquid interface inthe open fluidic system in the disclosed apparatus, the platformmaterial itself, though optional, does not need to be oxygen permeable.

Oxygenation Tail

A tail in addition to the main well for cell cultures is preferablydesigned for organs such as liver that higher levels of oxygenation forsurvival. The oxygenation tail has features supporting better diffusionand mixing of oxygen into the media such as shallow walls, fasterrecirculation, and independent inflow and outflow lines.

Exemplary layouts of the oxygenation tail includes a guiding groove tail(FIG. 33), a tail that is vertically rounded (e.g., and deepening), aflat tail with pinning columns, and a flat tail with meniscus pinninggroove tail.

The tail preferably includes a slanted surface such that the depth ofliquid can be as thin as 5 mm, 4 mm, 3 mm, 2 mm, or 1 mm, for sufficientaeration/oxygenation. In preferred embodiments, the apparatus supportscell culture survival for up to a month, two months, or longer.

In addition to the surface roughness and geometry of patterns on thetail surface, tortuosity of the tail as well as the width of the tailmay be modified to enhance oxygenation. For example, zig-zag shaped,tortuous tails provide a means to enhance oxygenation requiring areduced liquid volume for the liver module. Each turning loop or pointis where meniscus can pin to. FIG. 34 illustrates a zig-zag tail layoutfor the liver module. A total tail length of about 225 mm may support atotal tail volume of about 80 μL, for enhanced passive oxygenation,i.e., increasing the surface area of liquid exposed to air. FIG. 35illustrates a phase-guiding geometry that is repeatedly present alongthe oxygenation tail. This tail has alternating maximum width andminimum width, W₁ and W₂, respectively (W₁>W₂), in a repeated mannerthroughout the zigzag tail. Generally, within each segment of the tailbetween two U-shaped loops, there are two, three, four, or more repeatsof the alternating maximum and minimum width. The alternated widths eachhas its own length, e.g., every maximum width W₁ has a length of L₁, andevery minimum width W₂ has a length of L₂. Generally, L₁ is greater thanL₂ to accommodate more volume. The phase guiding feature is attributableto the angled increase of the width. As shown in FIG. 35, the angle, α,represents the increase of tail width relative to the forward directionof fluid flow in the tail. The angled increase of tail width, i.e., thenarrowing of tail width in the direction of the forward fluid flow,provides for better guidance for fluid directionality. W₁ may be in therange between 0.1 mm and 10 mm, for example between about 0.5 mm andabout 1 mm. W₂ may be in the range between 0.05 mm and 5 mm, for examplebetween about 0.3 mm and 0.5 mm. L₁ may be in the range between 0.5 mmand 10 mm. L₂ may be in the range between 0.1 mm and 10 mm. The angle,α, may be in the range of between 90° and 180°. In one embodiments, W₁is 0.8 mm, W₂ is 0.45 mm, L₁ is 1 mm, L₂ is 0.5 mm, and α is 150°. Thedepth of the zig-zag oxygenation tail may be a fixed depth or agradually varying depth in the range between about 0.05 mm and 5 cm, forexample between about 0.1 mm and about 10 mm. In one embodiment, thedepth of the tail is fixed at 0.5 mm.

Active Oxygenation Pumping Systems

Another means to enhance oxygenation is to utilize active oxygenationpumping system both ways between the liver well and the tail.

In some embodiments, the liver culturing well has within itself arecirculation pumping system, such that it has bottom-to-top flow ofoxygenated media. The oxygenation tail, generally containing liquid of ashallow depth, is recirculated within itself, such that the requiredoxygen concentration is reached in steady state. Active pumping allowsthe media from the well with scaffold (generally low on oxygen due tometabolic consumption) to be pumped to the oxygen-rich tail portion. Theoxygenated media from the tail is then pumped back to the well.

(5) Removable Insert

Removable Scaffold Integrable for Perfusion

Removable scaffolds may be used for MPS of choice, e.g., liver andpancreas, allowing off-platform seeding, manipulation, and assaying ofthe perfused tissues. Previous scaffolds by others are difficult toremove from the platform without causing damage or contamination. Insome embodiments, the removable scaffolds hold the filters and retainingrings that are a standard size, e.g., compatible with the disclosedplatform and/or commercially available LIVERCHIP®.

Scaffolds are configured to allow gentle insertion and removal viarotation and sliding along a sloped guide ramp. Some MPS compartmentsdesigned for use with these scaffolds include a sloped ramp to guide theinsertion and/or removal of the scaffold. A radial seal with theplatform is established with a low-binding o-ring (e.g., VITON® o-ring),allowing perfusion of the entire removable device.

FIG. 36 illustrates an exemplary modular, removable perfusion scaffoldthat allows perfused cell constructs to be gently removed from thesurrounding platform. The device includes a cup-like shell with radialo-ring seals 161 a/161 b and a flow-diffusing support structure at itsbase. Cells with or without biomaterials can grow on top of this support162. On the sides of the device in the upper body or the extension armof the scaffold, two holes allow for manipulation with sterile tweezersand small flanges help to guide it along a ramped thread. The ramp 159allows gentle insertion and removal via rotation, rather than verticalforce. Torqueing the scaffold into place minimizes the fluid pressureexperienced by the cells during insertion and removal.

Portability permits a number of functions that improve the usability ofa multi-well bioreactor. For example, constructs can be cultured inisolation, in unique cell media, and then selected for health andviability before they are joined together for a human-on-a-chipexperiment. A removable scaffold also allows complete isolation of onecell population from the multi-well bioreactor, allowing external assaysof cell health and metabolism to be performed without tainting theshared media with potentially harmful reagents.

The scaffold supports fluid inflow from below, i.e., the bottom surfaceof the well, and spillway outflow to other wells on the platform.

In some embodiment, a fluid aggregation device 163 is optionally addedon the removable, perfused scaffold to collect flow into a narroworifice. Fluid is mixed, and aggregated fluid is collected past a fixedlocation. At the outflow location, the oxygen tension or other fluidproperties can be queried by a small probe resting inside the top of thedevice. This way, sensors for average O₂ measurement do not requiredipping into the media or a part of the culture.

In some embodiments, a thin scaffold with a thin bottom/wall thicknessbetween about 0.05 mm and about 5 mm, preferably between about 0.1 mmand about 1 mm, or about 0.25 mm, situated on a membrane, is utilized toseed liver-associated cells for enhanced oxygenation, where the scaffoldis perforated with an array of channels (e.g., ˜0.3 mm diameter) and ismaintained in a re-circulating flow multi-well plate bioreactor. Livercells seeded into the scaffold form 3D tissue-like structures, which areperfused at flow rates sufficient to create a physiological oxygentension drop across the scaffold without excessive shear (Yates C, etal., Adv. Cancer Res. 97, 225-246 (2007)) and which can be maintained ina functional state for weeks in serum-free culture medium.

TRANSWELL®

The apparatus can contain wells that are compatible to hold multipleinsert vehicles for cell culture, such as commercially availableTRANSWELL® inserts or custom biomaterial scaffolds to support cells ororganoids.

(6) Moat to Reduce Evaporation

Additionally or alternatively, some embodiments of the apparatus includea humidity moat (element 104 in FIG. 5) to increase local humidity andreduce evaporation from the cell culture media. The moat may beconnected to external fluid source or fluid pumped in via build-in fluidchannels in the fluidic plate. Monitoring and pumping of fluid into themoat may be needed to compensate for loss of liquid due to evaporation,which is generally dependent on flow variations in the organ culturewells. The in-platform moats or micro evaporation chambers can be placedin any region of the fluidic plate to increase the moat area to minimizeevaporation from the wells, allowing for the creation of a humidmicroenvironment around the microphysiological well zones. Local heatingin the moats may also be used so most of the evaporation to maintain thehigh relative humidity above the platforms comes from the moats.

(7) Means for Addition or Withdrawal of Agent/Specimen

The apparatus may be connected to or used with one or more auto-samplingdevices. For example, the auto-sampling devices may be fluidicallyconnected to a low wetting sample collection tube.

(8) Pneumatic Actuation

On-board pumping saves dramatically on space and cost compared tocommercial syringe or peristaltic pumps, is more scalable, and allowsclosed-loop operation with very low dead volumes. Dynamic control offlow rates and directionality enables precise modulation ofconcentration profiles, allowing experimental operation to be scaled tomatch clinical/physiological distributions. Flow partitioning iscontrolled by imposing specific pumping frequency in the individualmicrophisiological systems, leading to specific flow-rates and;therefore, “partitioning” of flow.

Pneumatic Manifold/Plate

Pneumatically controlled fluid flow in the fluidic plate is generallyachieved via a three-chamber unit e.g., 220 a, 220 b, or 220 c of FIG.4. FIGS. 37A and 37B illustrate the details of an exemplarythree-chamber unit containing a pump in the center and two valves, eachon a side. When actuated sequentially, this valve arrangement canprovide directionality in flow by preventing backflow while allowing forforward fluid displacement. The well-characterized, reliablevalve-pump-valve units provide fixed strokes of fluid, which generatedeterministic fluid flow. This supports a broad, dynamic pumping rangebetween about 1 μL/day and about 10 mL/minute. In some embodiments, oneor more or all of the pumping channels have reversible flow, supportingpriming, sampling, and/or media/drug delivery configurations.

Generally, the pneumatic layer uses a pass-through design, whereair-conducting actuation lines with air inlets and air outlets 210 a and210 b (entry and exit being relative to the orientation of the plate)pass horizontally through the pneumatic plate, preferably in straightpaths. Straight paths of air-conducting actuation lines occupy less ofthe total platform footprint, and they support a faster pneumaticresponse (e.g., fast pressure change due to a low volume). Symmetricalair inlets and air outlets allow platforms to be daisy chained to runsimultaneously, connecting the outlets of one plate directly to the nextwith short lengths of tubing.

The pneumatic manifold generally employs a single bonded layer ofmaterial that allows for the creation of internal pneumatic channels.The pneumatic actuator membrane is generally a single layer polymericmaterial, e.g., polyurethane, that may be pressed between the pneumaticplate and the fluidic plate, or attached to one of the plates. Thefluidic side in this case contains fluidic channels with micron rangeresolution geometries that allow for direction and evacuation of fluid.Higher resolution of the fluidic channel generally leads to a slowerspeed of fluid movement, but it may allow for smaller death volumes.

4-Lane Dual-Channel Pump

In addition to the valve-pump-valve (V-P-V) pneumatic actuationconfiguration, a pump-pump-pump (P-P-P) configuration can be added toallow for a peristaltic movement of fluid.

Two or three sets of the three-chamber units may share one or twoair-conducting actuation lines, as shown in FIG. 38. When a fluidicchannel (of the fluidic plate) splits into two channels that arepneumatically regulated by both a set of V-P-V pump and a set of P-P-Ppump, which are placed one actuation line off and are 180° out-of-phase,the overall fluid combined from these two pulsating strokes has a smoothvolume profile. Four actuation lines for these two sets of pumpsaccounts for four degrees of freedom, which requires only one morepneumatic line than the V-P-V configuration.

One or more x-chamber units (x>=3) may be placed with one or moreair-conducting actuation lines off, in a similar principle to that shownin FIG. 38, to have a customized smoothness of flow volume.

Modular Pumping

Independent pumping allows for a different, e.g., higher, flow rate thanthat offered by the shared pumps. The incorporation of the fluid wellsinto the plate can reduce or eliminate the need for tubing, but the pumpdesigns can be amenable to driving external flows as well. Connections,such as ferrule connections, can be used to interface the built-in pumpswith external tubing, allowing a pumping manifold to drive a largenumber of flows simultaneously and in a compact package.

Pump Block for Single-Pass or Recirculation Perfusion

The top, or fluidic layer, contains the MPS compartments and the pumpsand channels that interconnect them. Below the fluidic layer, a thinmembrane such as a polyurethane membrane provides a sealing surface forthe channels and functions as the actuation layer for the pumps. Thebottom layer is a manifold (e.g., an acrylic manifold) that providespneumatic actuation of the pumps by routing compressed air to the baseof each pump chamber. When vacuum is applied, the membrane is pulleddown toward the pneumatic layer, filling the pump with fluid.Conversely, when pressure is applied, the membrane is forced up into thefluidic plate, driving fluid out of the pump. By actuating threechambers in series, a fixed displacement peristaltic pump is formed,allowing fluid to be moved linearly and against head pressure withoutbackflow (Domansky K, et al. Lab Chip 10(1):51-58 (2010); Walker I, etal. Journal of Micromechanics and Microengineering 17(5):891 (2007)).

Geometry to Reduce Membrane Stress

Different geometries of the pump other than one shown in FIG. 37B may beused. An alternative form includes designs where the horizontal channelsconnecting the pump to the valves has been removed, leaving only theV-shaped connection that directly links two adjacent chambers. Therational behind these V-geometries is that these features pneumaticallyisolate one chamber from the other when the membrane deflects such thatwhen one valve is actuated, its adjacent valve doesn't respond.Alternative configurations of pump geometry may reduce membrane stressand increase longevity of the actuation system and its consistency.

In some embodiments, further modifications to pump cavity geometries arecreated to render one concave contact and one convex contact between themembrane and the different V-shaped bridges, such that to preventmembrane deformation and breakage.

Validation of Pumping

Parity between the intended and actual flow rates enables well-mixingand intended molecular biodistribution among MPSs on a platform.Validation of the hardware may include direct measurements of pump ratesusing a capillary flow measurement tool. In some embodiments, the toolis interfaced with the outlets in each MPS compartment such that flowmay be measured as a function of time required to fill a fixed length oftubing. Deviations of flow rates from one fluidic plate to another maybe attributable to slight machining differences in the depth of the pumpchamber. Nevertheless, software calibration factors calculated from themeasurements may be entered to correct the pumping rates to within about±5%, ±4%, ±3%, ±2%, or ±1% of the target flow rates to adjust individualpumps. Generally, a small margin of error still allows for reliable anddeterministic operation, and accurate data interpretation.

(9) Means for Non-Contact Fluid Level Sensing

Capacitive Sensing with a Three-Electrode Design

The fluid level in a MPS well may be measured in a non-contacting mannerusing capacitance sensing. Electric charges go through plastic, suchthat probe can be placed next to the well but not in contact with themedia/culture of the well to avoid possible contamination. A capacitivesensing probe may be embedded in the wall material of wells (e.g., madefrom plastic). The circuit senses capacitance through the wall withoutfluid contact. Capacitive sensing electrodes sit behind the layer ofplastic isolated from fluid. As shown in FIG. 39, front electrodes 1201measure capacitance close to the fluid, while back electrodes 1203measure capacitance of plastic only (as reference). Front electrodes andback electrodes may be built on two sides of, therefore backed by, apolychlorinated biphenyl (PCB) board 1202 or a flex backing. The frontelectrodes have a sensing electrode in the middle and two referenceelectrodes, one on each side, which are coplanar to the central sensingelectrode (FIG. 40). This organization of reference electrodes and thesensing electrodes allows for good matching. Mirror opposing electrodesprovide self-guarding.

Previous designs places one negative (reference electrode) conductiveplate side-by-side and coplanar to the one positive (sensing electrode)in an attempt to measure liquid level from within the well wall in anon-contact manner. This causes the reference capacitor, Cref, to be inthe wrong place and results in inaccurate measurement of fluid level.

The apparatus utilizes an improved design containing three electrodes ofsymmetry, i.e., two reference electrodes coplanar to and symmetricalabout a central sensing electrode, coupled with a mirror set ofelectrodes on the back side of a PCB board. This results in Cref in theright place for self-guarding and better matching.

Optical Measurement

Light illumination of the fluid surface may indicate the depth of liquidin a well.

Pressure Sensing

In some implementations it may be advantageous to use a pressure sensorto determine the height of fluid in a well. This is possible given thewell-known relationship between pressure and height given by P=rho*g*h,where P is pressure, rho is fluid density, g is the acceleration ofgravity, and h is the fluid height. A pressure sensor of known types inthe art may be incorporated in fluidic connection to a well such thatthe pressure sensor is measuring pressure in the well at a knownreference height.

Feedback Control of Pumps for Volumes and Flow Rates

Measurements of a fluid level in a well can be transmitted to thecontrol unit that regulates the flow rates of liquid pumped into one ormore MPS wells. With a capacitive sensing measurement of the fluidheight in a well, the volume of liquid in that well can be calculatedwith a known surface are of the well. A real-time measurement of a fluidlevel therefore provides information of the volume flow rate (i.e.,difference in the fluid heights over the period of time). The feedbackfacilitates control of a set-point volume and pumping flow rate to MPSwells.

(10) Means for Temperature and Pressure Sensing and Control

Temperature is controlled by placing these platforms in an incubator,which maintains the global temperature within to 37±0.5 deg. Pressuresensing can be done with any of the static pressure sensing sensor typesknown in the art and give an indication of fluid height in the wells.Incorporating sensors to measure the well fluid height, using capacitivefluid level sensors or pressure sensors, in a feedback loop can help inactively controlling the well fluid volumes.

(11) Assembly of Integrated Components

Securing the Pneumatic Side with the Fluid Side

In some embodiments, bolting through alignment pins may be used as themeans to assemble the pneumatic side with the fluid side of thebioreactor. Insufficient sealing may result in fluid leakage.

Clamping may be used as an alternative means for securing the pneumaticside with the fluid side of the bioreactor. Mechanical, as well asmagnetic, clamps may be used to clamp the fluidic plate, the actuationmembrane, and the pneumatic plate together.

Whippletree pressure distribution mechanism may be utilized indistributing pressure across different air actuation lines or acrossplatforms.

Daisy Chain of Multiple Bioreactors

In some embodiments, two or more multi-organ MPS platforms are chainedone after another at the air inlets and outlets. With the pass-through,straight-path design of air-conducting actuation lines across thepneumatic plate, two or more platforms share pneumatics and a same setof controller. No additional hardware is needed to scale up the numberof platforms in a group. With symmetrical air inlets and outlets on eachbioreactor, daisy chaining is easy to set up and disassemble. Thisfeature saves time, cost, and space for operating severalbioreactors/MPS platforms at a same time.

Multilayered Organ-On-Chip Fluidic and Pneumatic Plates

FIG. 41 illustrates a criss-cross design of pumping system formultilayer stacking configurations of platforms.

Multilayered organ-on-chip plates may be assembled via internalchannels, made by either bonding of independent layers or 3D printing.The connections between pumps can overlap for this higher density offluidic plates. It is also compatible with different pumping and valvingconfigurations (e.g., such as those described in pneumatic actuation).The ability to have multilayered plates enables internal channels, whichmay replace the V-cuts in the valves, which reduces the pressure spikedue to valve operation and improves the performance for moredeterministic pumping profiles.

Multilayered plates may have several benefits over single-layer clampedplates. Higher density of pumps and channels generally allows for bettersealing, reduction of overall device footprint, no cleaning needed fordisposable manifolds, and an increased capability to multiplex controlswith crossing channels. It is also easier to divert channels aroundareas where imaging is desired, or where sensors need to be inserted formeasurement, in a multilayer plate configuration that the single-layerchain configuration. It provides more freedom in the layout of culturewells and the capability to incorporate new pump configurations.

External Connection

The multi-layer bioreactor apparatus may be connected to amicrocontroller and an external pneumatic solenoid manifold to provide asource of pumping from outside. For incubation of the bioreactorapparatus, a pneumatic solenoid manifold is connected that controls 36or a customized number of channels of tubing running through the back ofthe incubator to intermediary connectors. Inside the incubator, tubingis attached to the platform/bioreactor through valved breakawaycouplings to allow easy removal from the incubator for media changes andsampling. The connectors and software architecture allow the setup to becompatible with the 2-way, 3-way, 4-way, 5-way, 6-way, 7-way, or acustomizable number of multi-organ platforms, as well as many futureplatform variants, with minimal modification to the softwareconfiguration. Pump flow rates and calibration factors are set through agraphical user interface on a laptop, and can be sent to a customizedmicrocontroller (e.g., National Instruments myRIO-1900) over USB orWiFi. Both manual and pre-programmed control of pump rates are availabledepending on the experimental needs, and the microcontroller can runindependently of the laptop.

In some embodiments, a multi-organ MPS platform is connected to a localreservoir between controller and pump. In other embodiments, it isconnected to external microfluidic device for import of external supplyand export of waste.

Computerized Operation

Control software is configured to be instantiated/installed on or withan appropriate device, such a microcontroller (e.g., a NATIONALINSTRUMENTS MYRIO microcontroller) to allow continuous operation of aphysiomimetic platform without the need for a dedicated laptop ordesktop computer, although it is to be understood that some embodimentsmay utilize such a dedicated computer. The software operates thepneumatic pumps contained in the platform for the purposes of: fluidreplenishment and mixing (which provides nutrients and oxygen to thecells); introduction of fresh media from an internal or external source(feeding); removal of media to an internal or external collection vessel(sampling and waste collection); and/or dosing of test compounds, growthfactors, drugs, or other chemicals/proteins of experimental interest(dosing).

By providing a graphical user interface for the control of mixing,feeding, sampling, and dosing, this software facilitates the executionof complex experiments meant to replicate physiological interaction ofcompartmentalized organs.

The components and/or software also provide real-time feedback frompressure and vacuum sensors integrated into the hardware, which cancontain the microcontroller, pneumatic solenoids, pressure sensors,and/or power distribution electronics. In some embodiments, there isalso the capability to add an alarm for drift of pressure and vacuum outof acceptable ranges, and long-term data logging of these values can beimplemented.

In some embodiments, individual and global correction factors areincorporated in the software to allow correction for manufacturingvariability in pumps on the platform. For example, two pumps operatingat the same frequency (e.g., 2 Hz) will not always pump at exactly thesame rate if one is machined slightly deeper than the other. Bydetermining a correction factor (iteratively and/or experimentally), therate of the pump can be tuned to be very exact, where pumps weremeasured, calibrated, and re-measured to target 1 μL/s at 2 Hz. Thesoftware correction factors improve the performance of the pumps andminimize manufacturing variations across platforms.

In some embodiments, the microcontroller is WiFi compatible. Thesoftware can be configured with a web UI and backend (e.g., LabViewbackend) to control the pumps. This allows the user to access thecontrol panel of the software in a web browser without having to connectphysically to the microcontroller, allowing remote control andmonitoring of experiments from across the room or across the world.

An exemplary information flow from user to output includes thefollowing. A user accesses webUI over the local network or via VPNremotely. Control changes are passed from the WebUI to the backend,which adjusts the timing of the solenoid actuation to meet the desiredflow rates (accounting for individual and global pump calibrationfactors). The microcontroller then outputs a 3V digital on/off signal toa control board that amplifies that signal to a 12V analog actuation ofthe desired solenoids.

In some embodiments, there is a debug mode that allows manual operationof every single solenoid, for the purposes of finding malfunctioningsolenoids or manually opening/closing individual pumps and valves of theplatform.

Depending on different platform hardware design, the software isimplemented in a number of different ways, including: 4-way platformsoftware—controls pumping and calibration factors, displayspressure/vacuum data for 4 organ platform; 7-way platformsoftware—controls pumping and calibration factors, displayspressure/vacuum data for 7 organ platform, a program mode to defineautomated flow rate changes over time, and automated feeding andsampling controls from external ports on the platform; 3×GL platformsoftware—controls pumping and calibration factors, displayspressure/vacuum data for 3 organ platform, includes a program mode todefine automated flow rate changes over time, controls automated feedingand drug dosing (controlled volumes) to organs.

Also, a computer may have one or more input and output devices,including one or more displays as disclosed herein. These devices can beused, among other things, to present a user interface. Examples ofoutput devices that can be used to provide a user interface includeprinters or display screens for visual presentation of output andspeakers or other sound generating devices for audible presentation ofoutput. Examples of input devices that can be used for a user interfaceinclude keyboards, and pointing devices, such as mice, touch pads, anddigitizing tablets. As another example, a computer may receive inputinformation through speech recognition or in other audible format.

Such computers may be interconnected by one or more networks in anysuitable form, including a local area network or a wide area network,such as an enterprise network, and intelligent network (IN) or theInternet. Such networks may be based on any suitable technology and mayoperate according to any suitable protocol and may include wirelessnetworks, wired networks or fiber optic networks.

The various methods or processes outlined herein may be coded assoftware that is executable on one or more processors that employ anyone of a variety of operating systems or platforms. Additionally, suchsoftware may be written using any of a number of suitable programminglanguages and/or programming or scripting tools, and also may becompiled as executable machine language code or intermediate code thatis executed on a framework or virtual machine.

In some embodiments, the disclosed software to operate the multi-MPSplatforms is embodied as a computer readable storage medium (or multiplecomputer readable storage media) (e.g., a computer memory, one or morefloppy discs, compact discs, optical discs, magnetic tapes, flashmemories, circuit configurations in Field Programmable Gate Arrays orother semiconductor devices, or other non-transitory medium or tangiblecomputer storage medium) encoded with one or more programs that, whenexecuted on one or more computers or other processors, perform methodsthat implement the various embodiments of the invention discussed above.The computer readable medium or media can be transportable, such thatthe program or programs stored thereon can be loaded onto one or moredifferent computers or other processors to implement various aspects asdiscussed above.

Computer-executable instructions may be in many forms, such as programmodules, executed by one or more computers or other devices. Generally,program modules include routines, programs, objects, components, datastructures, etc. that perform particular tasks or implement particularabstract data types. Typically the functionality of the program modulesmay be combined or distributed as desired in various embodiments. Also,data structures may be stored in computer-readable media in any suitableform. For simplicity of illustration, data structures may be shown tohave fields that are related through location in the data structure.Such relationships may likewise be achieved by assigning storage for thefields with locations in a computer-readable medium that conveyrelationship between the fields. However, any suitable mechanism may beused to establish a relationship between information in fields of a datastructure, including through the use of pointers, tags or othermechanisms that establish relationship between data elements.

The acts performed as part of the method may be ordered in any suitableway. Accordingly, embodiments may be constructed in which acts areperformed in an order different than illustrated, which may includeperforming some acts simultaneously, even though shown as sequentialacts in illustrative embodiments.

III. Fabrication of Apparatus

The apparatus described above may be fabricated through molding,machining, and sterilization processes. A monolithic surfacemicromachined fluidic plate is preferred. It provides reliableperformance and it is easy to clean. All fluid contacting surfaces areaccessible for cleaning. All components have relatively long life time,and no delamination occurs in sterilization processes such as autoclave.Pneumatics can be easily cleared of condensation. Generally, theapparatus uses only two plate components bonded together, such that allpneumatic channels occupy the same plane within the plate. Inlets may bestacked by interleaving their channels and using drilled features toconnect the inlets at different vertical positions to the channel layer,thus packing them more densely on the side face of the manifold.

The turnaround cycle for modularized computer-aided design (CAD) andmachining is relatively quick. It is easy and rapidly customizableaccording to researcher's individual needs.

A. Materials

The organs-on-a-chip systems may be fabricated from polydimethylsiloxane(PDMS), polysulfone, and other materials. PDMS is a versatile elastomerthat is easy to mold (and thus highly amenable for prototyping), hasgood optical properties, and is oxygen permeable. In some embodiments,hydrophobic compounds including steroid hormones and many drugs exhibitstrong partitioning into PDMS, thus precluding quantitative analysis andcontrol of drug exposures (Toepke M W, et al., Lab Chip 6, 1484-1486(2006)).

In preferred embodiments, the fluidic plate is fabricated frompolysulfone (PSF). PSF is a rigid, amber colored, machinablethermoplastic with food grade FDA approval (21CFR177.1655) and USP ClassVI biocompatibility. It is generally resistant to a wide range ofchemical solvents, can be autoclaved, and is commonly used forinstrumentation and medical devices. PSF also has dramatically lowersurface adsorption and almost no bulk absorption of hydrophobic andlipophilic compounds (Ng S F, et al., Pharmaceutics 2, 209-223 (2010)).

All fluidic surfaces of the disclosed apparatus may be passivated priorto each experiment using serum albumin to further reduce the binding ofbiological molecules or drugs in the platform. The fluidic plate canalso be cleaned and reused as many times as needed.

The top fluidic plate may be machined from a monolithic block ofselected material, e.g., polysulfone (PSF) plastic, to includecompartments to accommodate each MPS and an interconnecting chamber(called mixer or mixing chamber) to integrate and mix return flows,representing systemic circulation. Microfluidic channels and pumps aremachined into the underside of the fluidic plate to convey fluid fromthe mixing chamber to each MPS. The individually addressable micro-pumpsare fabricated in-line with the built-in fluid channels, and may bebased on a 3-chamber, peristaltic pump-pump-pump design or avalve-pump-valve design. Additional pumps under each well providerecirculation flow, reducing nutrient and oxygen gradients within eachcompartment.

B. Techniques for Assembly and Bonding

The fluidic plate, pneumatic plate, and membrane (in sterilization bags)are generally assembled in a biosafety cabinet. Before assembly, asterile microplate lid is generally taped onto the fluidic plate toprotect the sterility of the cell culture region. The layers can then beassembled upside down to aid with visual alignment through the acrylicplate. Once the alignment pins mate with the fluidic plate, the platformcan be carefully removed from the hood, keeping pressure to maintain theseal between the plates. Screws can be inserted and tightened in anonsterile environment as long as the plates are not separated. Twofully assembled platforms can be daisy chained by connecting them withshort lengths of tubing connecting straight across to the correspondingports. Daisy chained platforms are most easily transported with a largemetal tray.

Platforms are assembled at a few days (e.g., 4 days) prior to the startof cell-culture experiment of interest. Surface passivation (priming) ofsterile platforms can be conducted with 1% BSA andpenicillin-streptomycin in PBS in volumes appropriate to eachcompartment. Pump function and tubing connections can generally bevisually confirmed by pumping from the mixer to each dry compartment,then by running the recirculation pumps backwards to clear all air fromthe channels. Spillways can be manually wetted with small volumes toensure spillway operation. Platforms are usually run overnight in theincubator to passivate and confirm full operation before the addition ofcells.

In some embodiments, fluidic plates are bonded to create closed fluidicpaths using a sintering method between plastic plates of specificpointedness.

FIGS. 42 and 43 illustrate a sintering method to bond multiplecomponents, where a bottom plate 250 has one or more small (e.g.,pointed) contact areas 251 a/251 b with a top plate 150 and some flatsurface areas 253 that are shorter in height by a difference of d₂ thanthe vertices of the small contact areas 251 a/251 b. Following force andtreatments to sinter the top plate with the bottom plate, previouslysmall surface areas are deformed and fused with the flat bottom surfaceof the top plate, to a height set by the joint height of the contactedflat surfaces 253, resulting in a fused component 350 having internalspace/volume 351 for passage of fluid.

In some embodiments, polyurethane (PU) membranes between about 20 and200 microns thick, preferably between 50 and 100 microns thick, such as50 microns thick, may be stretched on tension rings to maintain aconstant tension. They can be laser cut with the corresponding patternof screw holes on the pneumatic plate, if screw holes are present toalign the top plate with the pneumatic plate.

A membrane diaphragm (optionally containing elastomer in regionscorresponding to the pump and valve of the pneumatics) can be stretchedbetween the pneumatics plate and the plate for the fluidic culture, andpressed to adhere to the pneumatic plate. In some embodiments,automation is used to attach the membrane to the fluidic plate.

Alternatively, elastomer patches may be used on the membrane layer tocreate seals and hermetic pathways in fluidic plates. Elastomer materialmay be used only at regions of a membrane or a patch corresponding topneumatic pump and valves. Membranes containing elastomer patches can beprepared ahead of time and kept sterile for assembly of the chip. Thiswould facilitate the assembly and operation of organonchip plates wherean elastomer is deflected to create a pumping action only in verylocalized regions of the plate. A wide range of elastomer types andthicknesses may be applicable.

C. Surface Treatment to Control Wettability

Surface patterning may be used to control wettability of open fluidicpassages in the organ-on-chip plates. Machining patterns include zebra(linear), shark, concentric, and smooth surfaces.

The use of different machining processes and micro texturization candramatically affect wettability of culture plates for organs-on-chips.Surface finish may significantly modify polysulfone wettability up toabout a 40° change in the contact angle with water or a cell-culturemedia. Incubator conditions may also increases wettability to a slightextent of about 2-3° difference. It may be preferable for mesofluidicdevices to have an increased wettability in order to improve theperformance.

In general, dark polysulfone is more hydrophobic than light polysulfone.Selection of different grades of polysulfone provides another means tovary the wettability of the plates.

D. Sterilization

One or more sterilization procedures may be performed on thecell-culturing fluidic plate, the actuation membrane, and optionally thepneumatic plate. Sterilization techniques include gas treatment (e.g.,ethylene oxide), ionizing radiation, sonication, surface treatment(e.g., surfactant), and autoclave.

Generally before use, the top plate (e.g., polysulfone top plate) iscleaned and sterilized. First, the plate can be submerged in about 10%bleach for about 30 to 60 minutes, followed by a short rinse indistilled water. A residue-free surfactant was then used to remove anyremaining contaminants by sonicating, submerged in about 10% solution(e.g., 7× solution, MP Biomedicals #MP0976680HP) for about 15 minutes.Two subsequent 15-minute sonication cycles in fresh deionized waterfollow to remove all surfactant before a final deionized water rinse.The plate can then be air dried, sealed in a sterilization bag, andautoclaved.

Generally, the pneumatic plate does not require formal sterilization,but prior to assembly it may be wiped thoroughly with a kimwipe sprayedwith 70% ethanol to remove any dust or particles from the sealing areasthat contact the membrane.

Pneumatic actuator membranes may be rinsed in about 10% 7× solution andwith excess deionized water. Generally, an ethylene oxide gassterilization step follows after the membranes are air dried, and themembrane is allowed 24 hours to degas in a chemical fume hood.

E. Cells

Differentiated cell types and specialized cell types such as stem cellsand paneth cells, as well as microbiome for some embodiments such as gutMPS, may be added to the platform.

The microphysiological systems (MPSs) supported by the platform maycomprise primary cells, cell lines, pluripotent stem cells, progenitorcells, organoids, or any combination of mammalian or non-mammaliancells. For example, epithelial monolayers formed on transwell insertsfrom Caco2 cells or Caco-2 cells mixed with HT29 cells is one model ofthe gut, where circulation in the basal compartment (beneath thetranswell) serves to improve the mixing and thus transport of drugs andother agents from the apical side of the epithelial layer to the basalside of the epithelial layer; the mixing facilitates the rapiddistribution of drugs and other compounds in the basal compartment, andthus improves overall mixing between different MPS units on theplatform. This model can be made more sophisticated by adding a sourceof immune cells (e.g. dendritic cells or macrophages from humanperipheral blood monocytes or other sources) to the basal side of themembrane. It can be made even more sophisticated by culturing theepithelium on top of stroma encapsulated in an extracellular matrix gel;a similar arrangement can be used with primary intestinal cells. Asecond type of flow module is exemplified by the Liverchip-typearrangement, where flow is pumped through a scaffold containing 3Dtissues comprising multiple cell types on a scaffold designed todistribute flow through the tissue. In another configuration, a closedmicrofluidic device with flow channels on either side of a central gelregion may support tissues like 3D islets or lymph nodes, whereendothelial cells seeded into the gel with the islets or lymph nodesform 3D vessels that allow perfusion of the islets or lymph nodesthrough the channels. Islets or lymph nodes may also be maintained in agel in a transwell insert, and the basal side of the transwell insertcan be covered with endothelial cells. Finally, cells may be added tothe central circulation unit or any of the individual MPS circulationunits to allow cell trafficking. For example, PBMC added to the basalcompartment of the gut can traffic to the stroma across the membraneunder inflammatory signals.

In some embodiments where triple negative breast cancer (TNBC) (i.e.,lacking expression of estrogen, progesterone, and Her2 receptors)micrometastases in liver is modeled in the disclosed apparatus,MDA-MB-231 cells along with primary human hepatocytes andnon-parenchymal cells may be cultured.

In some embodiments where gut and liver MPS are modeled to assessinflammatory-related stimulation of dormant micrometastases, absorptiveenterocytes (e.g., CC2BB/e1 line) and mucin-secreting goblet cells(e.g., HT29-MTX line) may be seeded on the apical surface generally at anumber ratio between 20:1 and 5:1, more preferably between 13:1 and 7:1,or about 9:1; whereas dendritic cells, obtained from in vitrodifferentiation of human PBMCs-derived monocytes, may be seeded on thelower side of the membrane of a TRANSWELL® in one well of the apparatus.

Other cell lines or cell types may be added dependent on use.

IV. Applications

In vitro to in vivo translation (IVIVT) is an interpretive step thatcompares and validates MPS results to clinically-relevant outcomes. Thedisclosed apparatus may be applied with the IVIVT method in assessingadditional factors such as endogenous growth factor, inflammatory andhormone signals in the prediction of pharmacokinetics andpharmacodynamics (PK and PD). Compared with in vivo to in vitrocorrelation (IVIVC) and in vivo to in vitro extrapolation (IVIVE)methods in the prediction of PK, IVIVT goes a step further to includeanalysis of these additional factors and thus additionally predict PD,clinical toxicology, biomarkers, and patient stratification usinginformation from MPS technologies. Combined with physiologically-basedPK (PBPK) models for IVIVT, the disclosed apparatus provides an improvedquantitative forecast on human responses to test agents, taking intoaccounts missing organs, organ and media size mismatches, and drugexposure.

In some embodiments, the system can also be used to exemplify diseasesor disorders. For example, the apparatus may be used to establishmicro-metastases in the context of a relatively large (≧1 million cells)mass of liver cells, and then to analyze complex cell-cell communicationnetwork signatures using both measurements that can be routinely made inpatients (on the circulating medium) as well as measurements that cannotalso be made on patients—the kinetics of tumor cell growth and death.

A. Preclinical Drug Discovery

MPS supports survival and functional culture of one or more organs onthe chip for an extended period of time such as two, three, four, fiveweeks, two months, three months, or longer. Long-term multi-organcultures are particularly advantageous for studying the pharmacology oflow-clearance drugs, supporting repeated drug exposures, analyzingdrug-drug interactions, and modeling chronic diseases.

The platform can be used for target identification and validation,target-based screening, phenotypic screening, and other biotechnologicalapplications.

Cell and media volumes provide enough signal for commercial assays suchas ELISAs and high-content, multiplexed assays.

Multiple-omics measurements across the scales of information flow incells, from DNA to RNA to protein, protein activity states, andmetabolites, as well as similar types of analysis of patient-derivedimmune cell function.

Although standard culture systems are reasonably effective for mostsmall molecule drug PK assays, a vast number of diseases lackingadequate therapies have inflammation implications and are not wellrepresented or modeled in standard culture systems. The apparatus may beparticularly suitable for later stages of drug development thatgenerally involves the immune system. The apparatus has been shown torecapitulate a complex immunologically-based drug-drug interactionbetween the anti-IL6 receptor antibody, tocilizumab, and the metabolismof simvastatin—a phenomenon that could not be reproduced in standardcultures (Long T, et al., Drug Metab Dispos 44, 1940-1948 (2016)).

A wide range of drug agents (small molecules, peptide, proteins, nucleicacid, etc.) may be tested in the disclosed apparatus for medicinal,cosmetic, or scientific applications. Generally addition to the mixingwell mimicks an intravenous dosage, and addition to the gut well mimicksan oral dosage.

Agents are selected based on the disease or disorder to be treated orprevented.

B. Disease and Disorder to be Modeled

The multi-organ apparatus is a useful tool for disease modeling and drugdevelopment, especially in identifying and defining the appropriate“minimal set” of interacting organ systems to represent a disease state.

Drug development for a variety of diseases and/or disorders may beimproved utilizing the disclosed multi-organs on a chip apparatus byculturing relevant tissues or cell types for systemic studies. Complexindividual organs-on-chips that capture the local features of disease,especially inflammation, are preferably applicable for modeling systemicdiseases or diseases that are associated with multiple organs or involvemultiple types of cells. The diseases and/or disorders that may bemodeled in the disclosed bioreactor include but are not limited tocancers/tumors (e.g., tumors in the breast, bones, liver, lungs, andbrain), chronic inflammatory diseases (e.g. diabetes, arthritis,endometriosis, and Alzheimer's), non-malignant growth of endometriumoutside the uterus (endometriosis) or displaced into the uterine muscle(adenomyosis), abnormal liver functions such as those caused bynon-alcoholic fatty liver disease,

The system provides a means for exposing the cells to an agent todetermine its effect on the cells administering the agent in differentdosages, in a different dosing regimen, or in combination with one ormore other agents and determining its effect on the cells, as well aswherein the agent is administered to different cell types or cell typesassociated with one or more diseases or disorders. This allows one totest agents in vitro with human cells under conditions mimicking ahuman, at least in part, under controlled conditions, looking foreffects on other cell types, as well as on the cells one wants tomonitor for an effect. This is more rapid, more controlled, and yet notrestricted to a single class of cells or tissues.

EXAMPLES

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1. Perfused, Single-Organ Microphysiological Systems (MPSs) onthe Chip (1) Liver: Perfused, Coculture of Hepatocyte-Kupffer to ThreeWeeks Materials and Methods

Metabolic and immunologically competent 3D cryopreserved humanhepatocytes and kupffer cells were cocultured. Multiple hepatocyte andKupffer cell donors have been qualified in the MPS. Co-cultures wereresponsive to Lipopolysaccharide (LPS) stimulus down to 0.01 μg/ml.

Results

Table 1 shows the comparison of hepatocytes only and coculture ofhypatocyte and Kupffer cells at a 10:1 ratio over 7 days in a perfusedMPS platform.

TABLE 1 Biological function of liver cells vs. immune-competent liverMPS. Hepatocyte Hepatocyte + Function at Day 7 (n = 3) Only Kupffer(10:1) Albumin (μg/day/mg) 35 ± 11 53 ± 32 Urea (μg/day/mg) 175 ± 75 184 ± 25  CYP3A (pmol/min/mg) 2.9 ± 0.5 2.0 ± 0.7

The secretions of interleukin 6 (IL-6) and tumor necrosis factor alpha(TNFα) of the cocultured liver MPS were measured. The reproducibility ofIL-6 response to LPS stimulus was determined. A physiologically-relevant(relatively low) level of cortisol was used in the common media.Hydrocortisone (cortisol) enhanced differentiated function, butsuppressed inflammatory response.

The duration of cryopreserved human hepatocytes and kupffer cellco-cultures on the perfused MPS platform was extended to 3 weeks.

Expression of 84 hepatic genes remained stable between day 7 and 21.Kupffer cells remained inactivated for 21 days, until stimulated withLPS. Cell death marker LDH declined after seeding and remained at a lowconstant level. Hepatic phenotypic activity, including albumin andCYP450 remained measurable and superior to 2D cultures for 21 days.CYP450 activity was sensitive to hydrocortisone levels in the cultures.

(2) Lung: Primary Human Tracheobronchial Epithelium DifferentiationMaterials & Methods

A tracheobronchial module was developed in a TRANSWELL on the MPSplatform. Primary basal epithelial cells (all CK5+) were differentiatedat the air-liquid interface into a full subset of epithelial cell types.Different metrics were evaluated including transepithelial electricalresistance (TEER), mucus production, differentiated cell populations,and basal IL-8 production.

Results

Fluorescent microscopic images were taken and confirmed the expressionsof differentiation and functional markers: Tubulin (ciliated), Ck5(basal), Muc5Ac (goblet), and phalloidin (actin).

TABLE 2 Comparison of estimated and measured percentages ofdifferentiated cells on the lung MPS platform. Physiological MIT - DonorZ Cell Sub-type Estimates Lung MPS Basal    20%  28 ± 3% Goblet  1-5%  1± 0.5% Ciliated 30-50% 46 ± 11% Clara   <1% Not DeterminedTable 2 confirms the lung MPS model supported differentiation of cells,to a degree that aligned well with physiological estimates, which wasindicative of its function of primary human tracheobronchial epithelialmodel.

(3) Endometrium: Half-Primary Coculture of Epithelium-Stroma is Stableand Functionally Secretes Glycoprotein Materials & Methods

In a menstrual cycle, human endometrium undergoes a proliferative phase,marked by an increased level of estrogen, and a secretory phase, markedby an increased level of progesterone. In the secretory phase,endometrium secretes characteristic proteins such as glycodelin,prolactin, and insulin-like growth factor-binding protein 1 (IGF-BP1).

An exemplary endometrium model of cell culture system in a TRANSWELL® onthe MPS platform includes hydrogel encapsulating stromal cells andepithelial cells on top surface of the hydrogel were cultured on theapical side of the TRANSWELL®. The epithelial cell source was primaryhuman endometrial epithelial cells, which were readily obtained fromendometrial biopsies, had limited expansion and lifespan in culture,exhibited functional differences between harvest in proliferative andsecretory phase, and supported robust glycodelin secretion (secretoryphase cells). The cell line used was Ishikawa human stage 1adenocarincoma cell line, which were estrogen and progesterone receptorpositive, polarized in matrigel (Chitcholtan et al., Exp Cell Research,(2013)) or functionalized PEG gels, and had low/variable secretion ofglycodelin. The stromal cell source was primary human endometrialstromal cells, which were readily obtained from human biopsies, hadwell-established in vitro expansion protocols, and showed functionaldifference between harvest in proliferative and secretory phase. Thecell line used was human tert-immortalized cell line (tHESC), which washighly proliferative and stable, had low/variable secretion of prolactinor IGF-BP1 without strong decidualization cues, and could be quiesced inPEG gels.

Results

With Ishikawa epithelial cells, the apical medium contained estradioland progesterone. Ishikawa glycodelin secretion was below detectionlimit. Off-platform culture of tHESC had produced IGF-BP1 right at thedetection limit and a borderline detectable amount of prolactin (PRL)from primaries (likely due to a dilution effect). On-platform co-cultureof these “half-primary” cell lines were stable and had detectablefunctions from apical sampling.

(4) Gut/Immune: Coculture for Two Weeks Forming Intact Barrier, andDrug-Induced Leakiness Triggering Immune Response Materials & Methods

Physiological gut system features absorption and metabolism, intestinalimmune system, interactions between microbiome and mucosal interactions,immune interaction between cell and microbiome, and drug-immuneinteractions. An exemplary immune-competent gut model with cell culturein a TRANSWELL® on the MPS platform included enterocytes and gobletcells were cocultured on the apical side of the TRANSWELL and immunecells on basal side of the TRANSWELL membrane. The cell lines used wereCaco2 (enterocytes), HT29-MTX (goblet cells), and dendritic cells(immune cells), where enterocytes: goblet cells were cultured at a 9:1ratio (mimicking small intestine) to maturation off platform for 2 weeksand transferred to perfusion platform with added immune cells on thebasal side of the TRANSWELL membrane.

Results

When cultured off-platform (static medium), immune cells at 14 days hadmuch less survival than were cultured on-platform with basolateral flow,as confirmed via immunofluorescent microscopy. On-platform cultures at14 days supported the function of gut barrier cells and theirdifferentiation.

Example 2. Assessment of Drug Toxicity in Individual or 2-Way MPS on theChip (1) Liver/Immune: Toxicities of Diclofenac and Tolcapone

An immune-competent liver MPS model was prepared and studied. Diclofenacimpaired liver functions while cell death was minimal. Tolcaponedecreased mitochondrial activity and caused cell death.

(2) Gut/Immune: Toxicities of Diclofenac and Tolcapone

An immune-competent gut MPS model was prepared and studied. Diclofenacreduced epithelial barrier integrity, causing leaky gut with a minimalcell death. Tolcapone led to severe cellular death, hence a completeloss of epithelial function.

(3) Endometrium MPS: Toxicities of Diclofenac and Tolcapone

An endometrium MPS model was prepared and studied. Diclofenac-inducedloss of function correlating with cellular death. Tolcapone induced lossof function correlating with cellular death.

(4) Gut-Liver 2-Way: Administration of Tolcapone to Gut (“Oral”) Resultsin Gut-Specific Toxicity Materials & Methods

An immune-competent gut-liver interacted MPS model was prepared (detailssimilarly shown in Example 3) and studied. Tolcapone was added to thegut MPS to mimick “oral” administration. Metrics were an volume-weightedaverage from the 3 media compartments:Signal_(systemic)=Signal_(apical, gut)*V_(apical, gut)+Signal_(basal, gut)*V_(basal, gut)+Signal_(liver)*V_(liver).

Results

In the presence of tolcapone, gut and liver suffered MPS-specific lossof function, which was indicative of MPS-specific toxicity of tolcaponeeven delivered “orally”. Gut and liver also suffered from MPS-specificcell death markers whereas generic marker was insensitive to tolcapone,which indicated site of toxicity of tolcapone. Intestinal Fatty AcidBinding Protein (I-FABP) was used as a clinical biomarker of enterocytedamage for various disease states.

Example 3. Inflammatory Cytokine/Chemokine Crosstalk in Gut-Liver 2-WayMPS

Immune-competent human liver (hepatocytes and Kupffer cells) combinedwith intestinal (enterocyte, goblet cells, and dendritic cells)microphysiological systems is studied in this in vitro platform, toexamine gut-liver interactions under normal and inflammatory contexts.

The liver is situated downstream from the gut; as such, it is constantlyexposed to gut-derived factors, including metabolites, microbialantigens and inflammatory mediators. However, a quantitativeunderstanding of how these multicellular tissues communicate andcontribute to overall (patho)physiology is limited.

Background

Gut-liver crosstalk is an integral part of normal physiology and theirdysregulation is a common denominator in many disease conditions(Marshall J C, Host Defense Dysfunction in Trauma, Shock and Sepsis:Mechanisms and Therapeutic Approaches, eds Faist E, Meakins J L, &Schildberg F W (Springer Berlin Heidelberg, Berlin, Heidelberg), 243-252(1993)). Furthermore, gut and liver are major organs involved in drugabsorption and metabolism; changes to their functional interaction canimpact their response to therapeutic intervention (Morgan E T, DrugMetab Dispos 29(3):207-212 (2001); Deng X, et al., PharmacologicalReviews 61(3):262-282 (2009); Long T J, et al. Drug Metabolism andDisposition 44(12):1940-1948 (2016)). Gut and liver functions areintimately linked by virtue of their anatomical proximity. The liverreceives 70% of its blood supply from the gut via portal circulation; assuch, it is constantly exposed to gut-derived factors, includingmetabolites, microbial antigens and inflammatory mediators. Thegut-liver axis contributes considerably to the overall immunologicalstate of the body, with the gut being the largest immune organ and theliver harboring over 70% of the total macrophage population in the body.Interspecies differences often hinder the accurate prediction of humanresponses in animal models; the discrepancy is especially evident inprocesses involving the immune system (Mestas J, et al., The Journal ofImmunology 172(5):2731-2738 (2004); Giese C et al., Adv Drug Deliver Rev69:103-122 (2014)). For instance, few of the clinical trials for sepsistreatment have led to drug approval (Seok J, et al. Proc Natl Acad SciUSA 110(9):3507-3512 (2013). Fink M P Virulence 5(1):143-153 (2014)). Insepsis, gastrointestinal and hepatic injury have been associated withincreased disease severity (Rowlands B J, et al., British MedicalBulletin 55(1):196-211 (1999); Nesseler N, et al., Crit Care 16(5):235(2012)). Acute liver failure in the first 72 hours following onset ofsepsis was highly correlated with poor prognosis in septic patients.However, the lack of specific and predictive biomarkers precludes earlydiagnosis and patient stratification for effective intervention(Pierrakos C et al., Crit Care 14(1):R15 (2010)). Though the gut-liveraxis has been implicated in the escalation of a septic response, themechanisms and molecular players involved are poorly defined. Therefore,a fundamental understanding of gut-liver crosstalk is critical not onlyto the prediction of drug disposition, efficacy and toxicity, but alsothe elucidation of (patho)physiological mechanisms.

Materials & Methods

In vivo, the liver receives a dual blood supply, from the hepatic arteryand the portal vein (Liaskou E, et al., Mediators Inflamm 2012:949157(2012); Brown R P, et al., Toxicol Ind Health 13(4):407-484 (1997)).Correspondingly, the flow from the mixer well was partitioned into thegut and liver compartments to be 75% and 25%, respectively, scaledproportional to physiological cardiac output and hepatic blood flow, asshown below in

Table 3 and Table 4. Output from the gut module fed into the liver,representing portal circulation. A systemic recirculation flow rate of15 mL/day was used to ensure efficient distribution of exogenous andendogenous factors.

TABLE 3 Exemplary controlled flow rates in gut-liver MPS. CompartmentsFlow rates (μL/s) Mixer self-circ 1.0 mixer-gut 0.13 mixer-liver 0.043Liver self-circ 1.0 Gut self-circ, basal 0.25

TABLE 4 Exemplary controlled volume in gut-liver MPS. CompartmentsVolume (mL) Mixer 1.0 Liver 1.6 Gut Apical 0.5 Basal 1.5

The liver and gut tissue constructs in this study were multicellular and(innate) immune-competent, designed to encompass multiple key functions,including metabolic, barrier and immune functions. The liver microtissuecomprised a co-culture of human primary cryopreserved hepatocytes andKupffer cells at physiological 10:1 ratio, maintained in a culturemedium permissive for retention of inflammation responses, as previouslydescribed (Long T J, et al. Drug Metabolism and Disposition44(12):1940-1948 (2016); Sarkar U, et al. Drug Metabolism andDisposition 43(7):1091-1099 (2015)). The gut tissue was engineered tomimic the small intestine, with the epithelial monolayer derived from9:1 ratio of absorptive enterocytes (Caco2-BBE) and mucus-producinggoblet cells (HT29-MTX), and the immune compartment containing primarymonocyte-derived dendritic cells.

Human primary hepatocytes and Kupffer cells were purchased from LifeTechnologies (HMCPMS, HUKCCS). Scaffolds were washed 15 min in 70% EtOH,rinsed twice in PBS, incubated for 1 hour @RT in 30 ng/mL rat tailcollagen in PBS, left to dry overnight at room temperature, and punchedinto platforms (filter under scaffold under retaining ring). At day(−3)to experiment start, 10:1 ratio of hepatocytes and Kupffer cells werethawed into warm Cryopreserved Hepatocyte Recovery Medium (CHRM,Invitrogen), spun at 100 g for 8 min, and seeded at 6*10⁵ and 6*10⁴cells/well, respectively, in cold hepatocyte seeding medium (250 mLAdvanced DMEM+9 mL Gibco Cocktail A+12.5 mL FBS). After one day, themedia was changed to D(−2) medium (250 mL Advanced DMEM+10 mL CocktailB). After two more days, the medium was changed to common medium for theduration of the interaction experiment.

The common medium used in this study consisted of 500 mL Williams Emedium+20 mL Gibco Cocktail B+100 nM hydrocortisone+1%Penicillin-Streptomycin (P/S)).

Caco2 (clone: C2BBe1, ATCC, passage 48-58) and HT29-MTX (Sigma, passage20-30) cell lines were used for the intestinal epithelial cultures. Bothcell lines were passaged twice post thawing before their use forTRANSWELL seeding. Cell lines were maintained in DMEM (Gibco™ 11965-092)supplemented with 10% Fetal Bovine Serum (Atlanta Biologicals S11150,heat inactivated (HI) at 57° C. for 30 minutes), lx GlutaMax (Gibco™35050-061), 1× Non-Essential Amino Acids (Gibco™ 15140-148), and 1%Penicillin-Streptomycin (Gibco™ 15140-148). Caco2 at ˜70-80% confluenceand HT29-MTX at ˜80-90% confluence were harvested using 0.25%Trypsin-EDTA (Gibco™ 25200-056) and mechanically broken up into singlecells for TRANSWELL seeding. In seeding the cells into TRANSWELL®, theapical and basal side of TRANSWELL membrane were coated with 50 μg/mLCollagen Type I (Corning 354236) overnight at 4° C. The inserts werewashed with PBS−/− to remove unbound protein. 9:1 ratio of C2BBe1 toHT29-MTX was seeded onto 12-well 0.4 μm pore size TRANSWELL® inserts(Costar 3460) at a density of 10⁵ cells/cm². Seeding media contained 10%heat-inactivated FBS, 1× GlutaMax, 1% P/S in Advanced DMEM (Gibco™12491-015). The apical media was replaced 1 day post seeding to removeany unattached cells. The top and bottom compartments of the TRANSWELLplate are fed with 0.5 mL and 1.5 mL of seeding medium every 2-3 days.After 7 days, medium was switched to a serum-free gut medium byreplacing FBS with Insulin (5 μg/ml)-Transferrin (5 μg/ml)-SodiumSelenite (5 ng/ml) (Roche 11074547001).

To evaluate long-term functional viability in the gut-liver interaction,corresponding single tissue controls on platform were assayed withidentical media volumes, flow rates and flow partitioning. Allconditions were tested in a defined, serum-free common media thatsupported maintenance of gut and liver functions. The liver cells (10:1hepatocyte:Kupffer cell) were seeded on platform 3 days prior to thestart of the interaction experiment to allow for tissue formation andrecovery from seeding-related stress responses. The gut MPS wasdifferentiated for 3 weeks off-platform prior to the start of theinteraction experiment. During long-term operation, the common culturemedium in the system was replaced every 3 days.

To evaluate the health of the liver, samples from all compartments weretaken at every media change (every 72 hours) and assayed for albumin viaELISA (Bethyl Laboratories, E80-129).

Various Cytochrome P450 (CYP) enzyme activities were measured using adeveloped CYP cocktail assay (Pillai V C, et al., J Pharm Biomed Anal74:126-132 (2013)). Briefly, a cocktail of CYP substrates was added toliver compartment for a one hour incubation, and the supernatant wascollected for downstream processing. Substrate-specific metaboliteproduction was analyzed using mass spec.

Monocyte-derived dendritic cells were used as the immune component ofthe gut MPS. Briefly, peripheral blood mononuclear cells (PBMCs) wereprocessed from Leukopak (STEMCELL Technologies, 70500) and stored inliquid nitrogen. For each experiment, PBMCs were thawed and monocyteswere isolated using the EasySep Human Monocyte Enrichment Kit (STEMCELLTechnologies, 19058). The monocytes were differentiated to dendriticcell in Advanced RPMI medium (Gibco™ 12633-012) supplemented with 1×GlutaMax, 1% P/S, 50 ng/mL GM-CSF (Biolegend 572903), 35 ng/mL IL4(Biolegend 574004) and 10 nM Retinoic acid (Sigma R2625). After 7 daysof differentiation (at day 19-20 of gut epithelial cell maturation),immature dendritic cells were harvested using Accutase (Gibco™A11105-01) and seeded on to the basal side of the inverted gutTRANSWELLs® for 2 hours. After 2 hours, cells were returned to cultureplate and fed with gut media.

One-day post dendritic cell seeding, gut barrier function was assessed.Gut MPS with transepithelial electrical resistance values of at least250 Ohm*cm² were considered acceptable for experiment. For allinteraction experiments on platform, the gut MPS was maintained incommon media.

TEER measurement was performed using the EndOhm-12 chamber with an EVOM2meter (World Precision Instruments). The samples and the EndOhm chamberwere kept warm at ˜37° C. on a hot plate. Temperature was rigorouslymaintained during TEER measurement to minimize variability.

Secreted mucin was measured in apical gut compartment using an AlcianBlue assay. The mucin quantification protocol was adapted from (5).Briefly, media from apical was collected in low-binding tubes, and spundown at 10,000 g for 5 minutes, and the supernatant was collected andstored at −80° C. for subsequent analysis. Mucin secretion wasquantified against a standard of mucin (Sigma-Aldrich M3895) dissolvedin culture medium. Samples and standards were incubated in a 96-wellplate in a 3:1 mix of sample to Alcian Blue solution (Richard AllenScientific) for two hours. After incubation, plates were centrifuged at1640 g for 30 minutes at room temperature. Supernatant was removed byinverting the plates. Samples were rinsed twice with wash buffer (40%(v/v) of ethanol and 60% (v/v) of 0.1M sodium acetate buffer containing25 mM MgCl₂ at pH 5.8), with a 10-minute centrifugation step after eachrinse. After second spin, supernatant was removed and samples weredissolved with 10% SDS in distilled water. Plates typically requiredshaking or pipetting to fully resuspend samples. If bubbles formedduring resuspension, plates were centrifuged again for about 5 minutesprior to absorbance measurement on a Spectramax m3/m2e at 620 nm.

Cytokine levels were measured using multiplex cytokine assays, 37-plexhuman inflammation and 40-plex panel chemokine panels (Bio-RadLaboratories, Inc., Hercules, Calif., USA). Briefly, media samples werecollected in low-binding tubes, spun down at 10,000 g for 5 mins toremove cell debris, and the supernatant was stored in −80° C. Sampleswere measured at multiple dilutions to ensure the measurements werewithin the linear dynamic range of the assay. To minimize non-specificbinding to beads, bovine serum albumin (BSA) was added to achieve afinal concentration of 5 mg/mL in all samples. The protein standard wasreconstituted in the same media and the protein stock serially dilutedto generate an 8-point standard curve. Assays were run on a Bio-Plex 3DSuspension Array System (Bio-Rad Laboratories, Inc.). Data werecollected using the xPONENT for FLEXMAP 3D software, version 4.2(Luminex Corporation, Austin, Tex., USA). The concentration of eachanalyte was determined from a standard curve, which was generated byfitting a 5-parameter logistic regression of mean fluorescence on knownconcentrations of each analyte (Bio-Plex Manager software).

To obtain the total production amount per platform, the concentrationvalues were normalized by compartmental volume and added up across allcompartments (mixer, gut, liver) in each platform.

For both the baseline and inflamed conditions (at Day 3, n=4),intestinal and hepatic tissues were taken out of the platforms, and mRNAwas extracted using the PureLink RNA mini kit (ThermoFisher, 12183018A).Total RNA was analyzed and quantified using the Fragment Analyzer(Advanced Analytical), and cDNA was generated using the SMART-Seq v3 kit(Clontech). After cDNA fragmentation (Covaris S2), cDNA was end-repairedand adaptor-ligated using the SPRI-works Fragment Library System I(Beckman Coulter Genomics). Adaptor-ligated cDNA was then indexed duringPCR amplification, and the resulting libraries were quantified using theFragment Analyzer and qPCR before being sequenced on the Illumina HiSeq2000. 40-50 nt single-end read with an average depth of 15-20 million or5 million reads per sample were sequenced for the baseline and inflamedconditions respectively.

The FASTQ files were generated from the sequencing runs. The resultantreads were aligned to the human reference genome (GRch37/hg 19) usingTophat (version 2.0.12) (Kim D, et al. Genome Biol 14(4):R36 (2013)) toidentify reads that map to known transcripts, accounting for splicejunctions. HTSeq was used to determine the number of read countsuniquely overlap with known genomic features (Anders S, et al.,Bioinformatics 31(2):166-169 (2015)).

To identify significantly altered genes in isolation vs interactionconditions, differential gene analysis of count data was performed usingDESeq2 (Version 1.12.3) in R (Love M I, et al., Genome Biol 15(12):550(2014)). Only genes with greater than 1 cpm (count per million) in atleast 4 replicates, were included in the analysis. Multiple testingcorrection was performed using the procedure of Benjamini and Hochberg.Genes with an adjusted P-value below a FDR cutoff of 0.05 wereconsidered significant.

GOSeq R packages (Young M D, et al., Genome Biol 11(2):R14 (2010)) wasused to determine the over-represented biological of the differentiallyexpressed genes (FDR-adjusted P-values<0.05).

GSEA (version 2.2.3) was performed to identify differentially regulatedgene sets in isolation versus interaction, as describe in (SubramanianA, et al. (2005) Proceedings of the National Academy of Sciences102(43):15545-15550). To stabilize variance, the normalized count datawere processed using a regularized logarithm transformation in DESeq2.The signal-to-noise metric was used to generate the ranked list ofgenes. Canonical pathway gene sets from Molecular Signatures Database(c2.cp.v5.2) were used, which is a collection of curated genes sets frommultiple databases (e.g., Reactome, KEGG, BioCarta, PID). The empiricalP-values for each enrichment score were calculated relative to the nulldistribution of enrichment scores, which was computed via 1000 gene setpermutations. Gene sets with nominal P-value <0.01 and q-value <0.05were considered significant. Enrichment map (11), a Cytoscape plugin,was used to visualize the overlaps between significant gene sets and tofacilitate the systematic interpretation of the interdependencies amongdifferent biological processes.

Results

1. Baseline Liver- and Gut-Specific Functions were Maintained for aRelatively Long Term (>2 Weeks) in Gut-Liver Interactome.

Hepatic and intestinal functions assessed over two weeks of culture werecomparable for MPS maintained in communication or in isolation, asassessed by measurements of albumin production, gut barrier integrity,and gut mucus production. To evaluate liver metabolic function at theend of the 2-week experiment, the liver tissues from isolation andinteraction conditions (in the absence of gut) were dosed with acocktail of drug substrates targeting specific CYP450 enzymes.Drug-specific metabolite production in the media was measured using massspectrometry to determine the cytochrome P450 activity of the differentisoforms. Overall, the liver metabolic function was largely maintained,with modulation of select cytochrome P450 activities observed ingut-liver interaction. In particular, Cyp2C9 activity was significantlyenhanced, while Cyp3A4/5 activity was depressed. Gut-specific functions,including barrier integrity and mucus production, were not sizablyaltered between interaction and isolation controls. Subtle butsignificant modulation of cytochrome P450 activities (e.g., CYP3A4 andCYP2C9) were observed after 2 weeks of interaction.

2. Bile Acid Synthesis Pathway was Modulated in Bi-Directional Gut-LiveCrosstalk.

RNA sequencing was performed to profile the global transcriptomicchanges in the gut and liver tissues after 3 days of interaction, withcorresponding isolation controls (i.e., gut-only and liver-only). 105genes were significantly (FDR-adjusted P<0.05) altered in the liverduring interaction relative to isolation controls, of which 70 wereupregulated and 35 were downregulated. For the gut, 6 genes weresignificantly differentially expressed, of which 2 were upregulated and4 were downregulated. To understand the functional implications of thesemolecular changes, Gene Ontology (GO) analysis was performed to identifyoverrepresented biological processes that were altered underinteraction. Only significantly altered genes (FDR-adjusted P<0.05) wereused for GO analysis. The up-regulated biological processes in the liverprimarily involved cell division-related processes (Table 5).

TABLE 5 Biological processes up-regulated in liver under gut-liverinteraction. Adj. GO ID Biological Processes P-value P-value GO:0051302regulation of cell division 0.0E+00 0.0E+00 GO:0000070 mitotic sisterchromatid 0.0E+00 0.0E+00 segregation GO:0007059 chromosome segregation0.0E+00 0.0E+00 GO:0007049 cell cycle 9.6E−18 1.1E−14 GO:0006996organelle organization 7.3E−10 3.6E−07 GO:0008283 cell proliferation3.4E−09 1.4E−06 GO:0007017 microtubule-based process 4.9E−08 1.4E−05

Induction of cell cycle genes in liver may indicate an adaptive responseto gut-derived signals, although the soluble factors involved areunknown. On the other hand, the down-regulated biological processes inthe liver were mainly metabolic processes including bile acidbiosynthesis, lipid metabolism and xenobiotic metabolism (Table 6).

TABLE 6 Biological processes down-regulated in liver under baselinegut-liver interaction. Adj. GO ID Biological Processes P-value P-valueG0:0006694 steroid biosynthetic process 2.2E−05 1.5E−01 GO:0006579amino-acid betaine catabolic 2.8E−05 1.5E−01 process GO:0008202 steroidmetabolic process 4.7E−05 1.5E−01 GO:1901617 organic hydroxy compound1.0E−04 2.6E−01 biosynthetic process G0:0044283 small moleculebiosynthetic 1.8E−04 3.8E−01 process GO:0015914 phospholipid transport2.2E−04 3.9E−01 GO:0044281 small molecule metabolic process 3.3E−044.8E−01

Specifically, a mediator of the bile acid metabolism, CYP7A1, wasdown-regulated, which was indicative of a physiological coupling ofgut-liver functions, e.g., bile acide-mediated enterohepatic crosstalk.CYP7A1 is an enzyme central to bile acid synthesis; and its feedbackinhibition via FGF19 enterohepatic communication is well established(Ding L, et al., Acta Pharm Sin B 5(2):135-144 (2015)). The result onCYP7A1 was consistent with previous findings that perfusion ofprecision-cut rat intestinal and hepatic tissues in a microfluidicdevice for 7 hours resulted in bile acid-mediated CYP7A1 inhibition (vanMidwoud P M, et al., Lab Chip 10(20):2778-2786 (2010)). Though thenumber of significant genes in the gut samples was insufficient for GOanalysis, PCSK9, one of the differentially expressed genes, was found toplay a key role in cholesterol and lipid homeostasis. In fact,cholesterol and various types of bile acids have been shown tosuppresses PCSK9 mRNA expression in Caco2 intestinal cultures (LeblondF, et al. Am J Physiol Gastrointest Liver Physiol 296(4):G805-815(2009)). The studies showed the convergence on cholesterol and bile acidmetabolism pathways was indicative of transcriptional rewiring due tointer-MPS communication.

3. Coordinated Transcriptomic Changes and Tissue-Specific TranscriptomicChanges were Observed in Inflammatory Gut-Liver Crosstalk.

A large number of immune cells reside in the gut and liver duringhomeostasis and their activation in disease can contribute to systemicpathophysiology. Liver dysfunction associated with idiosyncratic adversedrug reactions has been linked to inappropriate immune activation(Cosgrove B D, et al. Toxicology and Applied Pharmacology 237(3):317-330(2009)). This study complemented parenchymal tissue models with immunecells in both the gut and liver to provide a morephysiologically-relevant culture platform for disease modeling and drugtesting. Reciprocal immune-epithelial cell communication drives systemicinflammation.

In an inflammatory context mimicking endotoxemia, 2 ng/mLlipopolysaccharide (LPS) was added in the circulating media from day 0(Gut MPS on platform) to day 3 (RNAseq was performed) while theoperation of the system and the isolation controls were tested in asimilar way to the baseline studies above. The LPS concentration waschosen based on clinically-relevant range of plasma endotoxin (2-10ng/mL) reported in patients with inflammatory diseases (Guo S, et al, AmJ Pathol 182(2):375-387 (2013)).

RNA sequencing was performed to assess the global molecular changesassociated with inflammatory gut-liver crosstalk. For the liver, 2548genes were significantly altered in the interaction, of which 1137 geneswere upregulated and 1411 genes were downregulated. GO analysis of thedifferentially expressed genes showed upregulation of cytokine responseand antigen processing and presentation pathways, and downregulation oflipid and xenobiotic metabolism pathways (Table 7).

TABLE 7 Biological processes up-regulated in liver under inflammatorygut-liver interaction. Adj. GO ID Biological Processes P-value P-valueGO:0006955 immune response 1.7E−28 2.3E−24 GO:0006952 defense response1.5E−27 1.0E−23 GO:0019221 cytokine-mediated 2.5E−25 4.6E−22 signalingpathway GO:0060337 type I interferon 2.0E−25 4.4E−22 signaling pathwayGO:0051707 response to other organism 8.8E−22 7.6E−19 GO:0019882 antigenprocessing and presentation 3.1E−06 2.9E−04 GO:0002250 adaptive immuneresponse 1.0E−07 1.2E−05 . . . see further list in Table 11 and below.

TABLE 8 Biological processes down-regulated in liver under inflammatorygut-liver interaction: Adj. GO ID Biological Processes P-value P-valueGO:0044281 small molecule metabolic process 7.8E−97 1.0E−92 GO:0006082organic acid metabolic process 5.4E−78 1.8E−74 GO:0055114oxidation-reduction process 5.4E−69 1.4E−65 GO:0044710 single-organismmetabolic process 2.3E−56 5.0E−53 GO:0032787 monocarboxylic acid 4.0E−547.4E−51 metabolic process GO:0006629 lipid metabolic process 7.4E−539.7E−50 GO:0006805 xenobiotic metabolic process 1.3E−23 5.7E−21 . . .see further list in Table 12 and below.

For the gut, 780 genes were significantly altered during interaction, ofwhich 290 genes were upregulated and 490 genes were downregulated.Similarly, GO analysis revealed upregulation of defense response,antigen processing and presentation pathways and protein translation;down-regulated pathways included alcohol biosynthesis, steroid and lipidmetabolism (Table 9).

TABLE 9 Biological processes up-regulated in gut under inflammatorygut-liver interaction. Adj. GO ID Biological Processes P-value P-valueGO:0006952 defense response 4.5E−20 5.3E−16 GO:0060337 type I interferonsignaling pathway 1.2E−19 5.3E−16 GO:0002376 immune system process1.4E−13 2.2E−10 GO:0034097 response to cytokine 9.2E−13 1.1E−09GO:0006082 organic acid metabolic process 9.8E−10 3.5E−07 GO:0019882antigen processing and presentation 6.0E−07 1.4E−04 tRNA aminoacylationfor protein GO:0006418 translation 6.6E−10 2.7E−07 . . . see furtherlist in Table 11 and below.

TABLE 10 Biological processes down-regulated in gut under inflammatorygut-liver interaction. Adj. GO ID Biological Processes P-value P-valueGO:0046165 alcohol biosynthetic process 5.5 × 10⁻¹⁶ 7.0 × 10⁻¹²GO:0008202 steroid metabolic process 2.6 × 10⁻¹⁴ 1.0 × 10⁻¹⁰ organichydroxy compound G0:1901615 metabolic process 3.2 × 10⁻¹⁴ 1.0 × 10⁻¹⁰GO:0044281 small molecule 4.4 × 10⁻¹² 4.3 × 10⁻⁰⁹ metabolic processGO:0032787 monocarboxylic acid 4.8 × 10⁻¹¹ 3.8 × 10⁻⁰⁸ metabolic processGO:0006629 lipid metabolic process 7.8 × 10⁻¹¹ 5.9 × 10⁻⁰⁸ GO:0055114oxidation-reduction process 5.0 × 10⁻⁰⁸ 2.4 × 10⁻⁰⁵ . . . see furtherlist in Table 12 and below.

In addition to gene-based GO analysis that focused only on thesignificantly altered genes determined by an arbitrary statisticalcut-off, Gene Set Enrichment Analysis (GSEA) was also performed touncover coordinated changes in groups of genes that are functionallyrelated. GSEA can reveal more nuanced pathway regulation that might havebeen masked by strict cut-offs in gene-based approach. Generally, GSEAresults were largely consistent with GO analysis outcomes, but withgreater interpretability and generality. Consensus clusters of gene setsfrom different databases were obtained, which contained overlapping butdistinct groups of genes that define major biological processes.Specifically, inflammation-related pathways centered around IFNα/β/γsignaling were up-regulated whereas metabolic processes involvingcholesterol and lipid metabolism were down-regulated in both the gut andliver in interaction (Table 11). The pronounced alteration ininflammatory processes and lipid metabolism was characteristic of asepsis response.

TABLE 11 Gene sets commonly up-regulated in both gut and liver in thegut-liver MPS Pathways Liver: q-val Gut: q-val IFNReactome_Interferon_alpha_beta_signaling 0.0 + 00 0.0 + 00 signalingReactome_Interferon _gamma_signaling 0.0 + 00 0.0 + 00Reactome_Interferon _signaling 0.0 + 00 0.0 + 00 CytokineReactome_cytokine_signaling_in_immune_system 0.0 + 00 3.0 × 10⁻⁰³Antigen Kegg_antigen_processing_and_presentation 1.0 × 10⁻⁰³ 1.0 × 10⁻⁰³processing Reactome_antigen_presentation_folding_assembly_(—) 2.0 ×10⁻⁰³ 30 × 10⁻⁰³ and peptide loading of class

 MHC Reactome_antigen_processing_cross_presentation 6.0 × 10⁻⁰³ 2.5 ×10⁻⁰² Reactome_ER_phagosome_pathway  7.0 × 10−03 4.0 × 10⁻⁰³ ImmuneKegg_intestinal_immune_network_for_IGA_production 1.8 × 10⁻⁰² 2.5 ×10⁻⁰² processes Reactome_immunoregulatory_interactions_(—) 4.0 × 10⁻⁰³2.4 × 10⁻⁰² between a lymphoid and a non lymphoid cellKegg_allograft_rejection 1.0 × 10⁻⁰³ 0.0 + 00Kegg_autoimmune_thyroid_disease 2.0 × 10⁻⁰³ 0.0 + 00Kegg_viral_myocarditis 2.0 × 10⁻⁰³ 4.0 × 10⁻⁰³Kegg_graft_versus_host_disease 3.0 × 10⁻⁰³ 0.0 + 00Kegg_Type_I_diabetes_mellitus 7.0 × 10⁻⁰³ 0.0 + 00

indicates data missing or illegible when filed

TABLE 12 Gene sets commonly down-regulated in both gut and liver in thegut-liver MPS. Pathways Liver: q-val Gut: q-val EndogeneousReactome_cytochrome_p450_arranged_by_substrate_type 0.0 + 00 3.3 × 10⁻⁰²and xenobiotic Reactome_phase I_functionalization_of_compounds 0.0 + 004.4 × 10⁻⁰²

Kegg_metabolism_of_xenobiotics_by_cytochrome_p450 0.0 + 00 4.6 × 10⁻⁰²Lipid Kegg_PPAR_signaling_pathway 0.0 + 00 4.0 × 10⁻⁰³ metabolismReactome_lipid_digestion_mobilization_and_transport 4.0 × 10⁻⁰³ 3.9 ×10⁻⁰² Reactome_lipoprotein_metabolism 1.0 × 10⁻⁰² 4.3 × 10⁻⁰²Reactome_metabolism_of_lipids_and_lipoproteins 1.0 × 10⁻⁰³ 8.0 × 10⁻⁰³Steroid and Kegg_steroid_hormone_biosynthesis 1.1 × 10⁻⁰² 0.0 + 00 bileacid Reactome_bile_acid_and_bile_salt_metabolism 0.0 + 00 3.2 × 10⁻⁰²metabolism

indicates data missing or illegible when filed

In addition to the co-modulated pathways, tissue-specific regulation wasalso identified. Pathways involved in hypoxia and TGFβ/SMAD signalingwere exclusively upregulated in the liver in interaction, suggestive ofa pro-fibrotic response. Although the current study focused on acuteinflammation, chronic liver inflammation has been linked to liverfibrosis.

In the gut, PI3K-mediated ERBB2 and ERBB4 signaling was upregulated,which was indicative of a wound healing or anti-apoptotic response,possibly serving as a protective mechanism. Previously, ERBB2 (YamaokaT, et al. Proc Natl Acad Sci USA 105(33):11772-11777 (2008); Zhang Y, etal., Lab Invest 92(3):437-450 (2012)) and ERBB4 (Frey M R, et al.,Gastroenterology 136(1):217-226 (2009)) signaling have been shown, invitro and in vivo, to protect against TNF-induced apoptosis inintestinal epithelial cells and provide pro-survival and pro-healingeffects following intestinal injury.

Complete lists of gene sets involved in tissue-specific modulation areshown below:

Gene sets up-regulated uniquely in liver during inflammatory gut-livercrosstalk included Biocarta_TNFR2_pathway,St_tumor_necrosis_factor_pathway, PID_TNF_pathway,Reactome_chemokine_receptors_bind_chemokines,Kegg_cytokine_cytokine_receptor_interaction,Kegg_rig_i_like_receptor_signaling_pathway,Kegg_cytosolic_dna_sensing_pathway,Reactome_negative_regulators_of_rig_i_MDA5_signaling,Naba_secreted_factors, PID_CD40_pathway, PID_hif1_tfpathway,PID_hif2pathway, PID_i123_pathway, Kegg_primary_immunodeficiency,Reactome_antiviral_mechanism_by_ifn_stimulated_genes,Reactome_)O.o_linked_glycosylation_of_mucins,Reactome_regulation_of_hypoxia_inducible_factor_hif_by_oxygen,Reactome_rig_i_mda5_mediated_induction_of_ifn_alpha_beta_pathways,Reactome_signaling_by_tgf_beta_receptor_complex,Reactome_smad2_smad3_smad4_heterotrimer_regulates_transcription,Reactome_traf6_mediated_irf7_activation,Reactome_transcriptional_activity_of_smad2_smad3_smad4_heterotrimer, andSt_fas_signaling_pathway.

Gene sets up-regulated uniquely in gut during inflammatory gut-livercrosstalk included PID_IL12_2pathway, Kegg_abc_transporters,Reactome_amino_acid_synthesis_and_interconversion_transamination,Kegg_aminoacyl_trna_biosynthesis,Reactome_cytosolic_trna_aminoacylation, Reactome_trna_aminoacylation,Kegg_cell_adhesion_molecules_cams, Kegg_histidine_metabolism,Reactome_activation_of_genes_by_atf4,Reactome_perk_regulated_gene_expression,Reactome_PI3K_events_in_erbb2_signaling, andReactome_PI3K_events_in_erbb4_signaling.

Gene sets down-regulated uniquely in liver during inflammatory gut-livercrosstalk included Biocarta_ami_pathway, Biocarta_intrinsic_pathway,Kegg_alanine_aspartate_and_glutamate_metabolism,Kegg_arachidonic_acid_metabolism, Kegg_arginine_and_proline_metabolism,Kegg_beta_alanine_metabolism,Kegg_biosynthesis_of_unsaturated_fatty_acids, Kegg_butanoate_metabolism,Kegg_citrate_cycle_tca_cycle, Kegg_complement_and_coagulation_cascades,Kegg_drug_metabolism_cytochrome_p450,Kegg_drug_metabolism_other_enzymes, Kegg_fatty_acid_metabolism,Kegg_glycine_serine_and_threonine_metabolism,Kegg_glycolysis_gluconeogenesis, Kegg_ropanoate_metabolism,Keggglyoxylate_and_dicarboxylate_metabolism, Kegg_histidine_metabolism,Kegg_linoleic_acid_metabolism, Kegg_lysine_degradation,Kegg_oxidative_phosphorylation, Kegg_parkinsons_disease,Kegg_peroxisome, Kegg_proximal_tubule_bicarbonate_reclamation,Kegg_pyruvate_metabolism, Kegg_retinol_metabolism,Kegg_tryptophan_metabolism, Kegg_tyrosine_metabolism,Kegg_valine_leucine_and_isoleucine_degradation, PID_hnf3b_pathway,Reactome_biological_oxidations,Reactome_branched_chain_amino_acid_catabolism,Reactome_citric_acid_cycle_tca_cycle,Reactome_fatty_acid_triacylglycerol_and_ketone_body_metabolism,Reactome_formation_of_fibrin_clot_clotting_cascade,Reactome_metabolism_of_amino_acids_and_derivatives,Reactome_peroxisomal_lipid_metabolism, Reactome_phase_ii_conjugation,Reactome_pyruvate_metabolism_and_citric_acid_tca_cycle,Reactome_respiratory_electron_transport,Reactome_respiratory_electron_transport_atp_synthesis_by_chemiosmotic_coupling_and_heat_production_by_uncoupling_proteins_,Reactome_synthesis_of_bile_acids_and_bile_salts,Reactome_synthesis_of_bile_acids_and_bile_salts_via_7alpha_hydroxycholesterol, and Reactome_tca_cycle_and_respiratory_electron_transport.

Gene sets down-regulated uniquely in gut during inflammatory gut-livercrosstalk included Biocarta_TNFR2_pathway, Kegg_DNA_replication,Kegg_pantothenate_and_coa_biosynthesis,Kegg_pentose_and_glucuronate_interconversions,Kegg_steroid_biosynthesis, Kegg_terpenoid_backbone_biosynthesis,PID_aurora_b_pathway, PID_hif1_tfpathway,Reactome_activation_of_atr_in_response_to_replication_stress,Reactome_activation_of_the_pre_replicative_complex,Reactome_cholesterol_biosynthesis,Reactome_deposition_of_new_cenpa_containg_nucleosomes_at_the_centromere, Reactome_DNA_strand_elongation,Reactome_e2f_mediated_regulation_of_dna_replication,Reactome_fatty_acyl_coa_biosynthesis,Reactome_formation_of_tubulin_folding_intermediates_by_cct_tric,Reactome_g1_s_specific_transcription, Reactome_g2_m_checkpoints,Reactome_transport_of_vitamins_nucleosides_and_related_molecules, andReactome_triglyceride_biosynthesis.

4. Systemic Inflammation Suppressed Hepatic Detoxification Function.

Hepatic clearance of endogenous and xenobiotic compounds is mediated bytwo mechanisms, i.e., metabolism and bile elimination. The resultsrevealed inflammatory crosstalk negatively affected both of thesepathways and might lead to the buildup of toxic by-products.Collectively, CYP1A2, CYP2C9, CYP2C19, CYP2D6, an CYP3A4 and 3A5 areresponsible for the metabolism of over 90% of known drugs (Jacob A, etal., Int J Clin Exp Med 2(3):203-211 (2009); Ebrahimkhani M R, et al.,Adv Drug Deliv Rev 69-70:132-157 (2014)). All of these were suppressedin the liver in the integrated system, likely due to accumulation ofinflammatory mediators, such as IL6, TNFα, and/or type I interferons(Long T J, et al. Drug Metabolism and Disposition 44(12):1940-1948(2016); Huang S M, et al. Clin Pharmacol Ther 87(4):497-503 (2010)).

In short, lipid metabolism and inflammation were the dominant pathwaysaltered during gut-liver interaction. Lipoprotein binding to LPS canredirect the LPS uptake from Kupffer cells to hepatocytes, therebyattenuating immune activation and facilitating bile clearance of LPS(Khovidhunkit W, et al., J Lipid Res 45(7):1169-1196 (2004)). Peroxisomeproliferator-activated receptors (PPARs), master regulators of lipidmetabolism, have been shown to exert anti-inflammatory effects (Varga T,et al., Biochim Biophys Acta 1812(8):1007-1022 (2011)). Taken together,the suppression of apolipoprotein synthesis and PPAR signaling observedduring inflammatory gut-liver crosstalk indicates a potential loss of aprotective mechanism, thereby intensifying inflammation in immune andepithelial cells. The complexities in systemic response to perturbationsmotivate the need for multi-cellular and multi-organ experimentalmodels.

Sepsis patients are susceptible to adverse drug reactions due toinflammation-induced suppression of liver metabolic function,specifically the activity of cytochrome P450 enzyme system (Kim T H, etal., Febs J 278(13):2307-2317 (2011)). The results demonstrated alteredmRNA expression of Phase I and Phase II metabolic enzyme in inflammatorygut-liver crosstalk. Thus, accurate prediction of drug pharmacokineticsand pharmacodynamics necessitates the consideration for multi-organinteraction as well as the physiological context (ie., health vs.disease). This is especially pertinent for drugs with a narrowtherapeutic window because even modest changes to cytochrome P450activities can precipitate toxicity.

5. Cytokine Levels in the Gut-Liver Integrated System Deviates from theLinear Sum of Individual, Isolated Systems.

The levels of secreted cytokines and chemokines were measured in themedia at 6, 24, and 72 hours post stimulation to examine the temporalevolution of the inflammatory response. Pairwise hierarchical clusteringwas performed on the 72 hr cytokine measurement to explore thecorrelations of cytokine responses among the analytes and conditions.Unsupervised principal component analysis (PCA) revealed that the over96% of the covariance in the cytokine dataset can be captured by thefirst 2 principal components. PC1 accounted for 76.5% of the variabilityin the data, segregating the interaction versus isolation controls; PC2accounted for 19.8% of the total variability and discriminated the gutand liver only conditions. The loading plot depicted the relativecontribution of each analyte to the 1^(st) and 2^(nd) principalcomponents. All analytes were positively loaded on PC1 and contributedto the cytokine level in the integrated system, whereas loadings on PC2can help infer the primary tissues of origin of the circulatingcytokines/chemokines in the integrated system. While none of the solublefactors were unique to gut or liver, multivariate cytokine patterns canreveal tissue-specific signatures.

In order to accurately assess the contribution of inter-MPS crosstalk tothe integrated inflammatory response, the measured cytokine levels inthe interacting system were compared to the theoretical linear sum ofthe isolated conditions. The cytokine level observed in isolationaccounted for cytokine output due to direct TLR4 activation andintra-MPS paracrine signaling. The actual (measured) cytokine levels inthe integrated systems deviated significantly from the linear sum of theisolated systems, revealing non-linear modulation of cytokine productionas a result of inter-MPS communication. Approximately 58% of theanalytes were linearly additive, 23% were less than additive, and 19%were more than additive, some very markedly so. Interestingly, severalcytokines exhibited similar temporal dynamics as CXCL6, which waslinearly additive up to 24 hr, and then diverged from linear sum andbecame more than additive. This may suggest a threshold-dependentregulation, where cytokine production is dependent on the accumulationof upstream inducer molecules during organ crosstalk.

6. Inflammatory-Related CXCR3 Ligand was Greatly Amplified in Gut-LiverInteraction.

Table 12 showed a notable more than additive amplification of CXCR3ligands, where CXCL10 (IP10) and CXCL11 (I-TAC) were most significantlymore than additive and CXCL9 (MIG) was borderline significant. Thefractions of total analytes that were additive, subadditive, and morethan additive in terms of the level in the gut-liver MPS, compared tothe linear sum of the levels in individual gut and individual liver,were 58%, 23%, and 19%, respectively. CXCR3 signaling has beenimplicated in autoimmunity, transplant rejection, infection, and cancer(Groom J R, et al., Immunol Cell Biol 89(2):207-215 (2011); Singh U P,et al., Endocr Metab Immune Disord Drug Targets 7(2):111-123 (2007)).

TABLE 12 Cytokines/chemokines statistically different from linear sum(Adj. P-value < 0.05) and the corresponding receptors.Cytokines/chemokines Receptors Target cells Sub- CCL21 CCR7, CCR11thymocytes & activated T cells additive CCL1 CCR8, CCR11 monocytes, NKcell, B cells & DCs CCL11 CCR3 leukocytes, eosinophils CXCL12 CXCR4,CXCR7 lymphocytes, endothelial progenitors CHI3L1 — — CCL22 CCR4lymphocytes, monocytes, DCs, NK cells MIF CXCR2, CXCR4 most hematopoeticcells & endothelial cells IFN-γ IFNγ-R immune cells & epithelial cellsCCL27 CCR10 memory T lymphocytes CXCL13 CXCR5 B lymphocytes SynergisticCXCL10 CXCR3 Th 1 cells, NK cells CXCL11 CXCR3, CXCR7 Th 1 cells, NKcells, monocytes, neutrophils CXCL6 CXCR1, CXCR2 neutrophils CCL20 CCR6lumphocytes, DCs CCL2 CCR2 monocytes, basophils CX3CL1 CX3CR1 leukocytesCCL19 CCR7 lymphocytes, DCs, hematopoetic progenitors CXCL9 CXCR3 Th1cells, NK cells

These results showed that consideration of gut-liver crosstalk may beimportant for assessing systemic inflammatory processes and theirpotential influence on disease development.

RNA sequencing data showed activation of IFNα/β/γ signaling pathways inboth the gut and liver during organ crosstalk. TNFα can magnifyIFN-dependent production of CXCR3 ligands. PCA loadings revealed thatTNFα was predominately gut-derived and IFNγ was produced at comparablelevels by both the gut and the liver. It was plausible that gut(dendritic cells)-derived TNFα interacted with tissue-specific IFNγsignaling to drive CXCR3 ligand production in both the gut and liver.However, the relative contribution of epithelial and immune compartmentto the integrated response was difficult to ascertain. Although immunecells are the principal responders to endotoxin due to higher expressionof TLR4 as shown in Table epithelial cells also contribute toinflammation indirectly via activation by immune cell-derived cytokines,such as TNFα and IL-1 (Nguyen T V, et al. Drug Metab Dispos43(5):774-785 (2015); Yeruva S, et al., Int J Colorectal Dis23(3):305-317 (2008); Dwinell M B, et al., Gastroenterology 120(1):49-59(2001)).

TABLE 14 TLR expression (Log10, normalized to GAPDH) Cell types TLR1TLR2 TLR3 TLR4 TLR5 Primary human hepatocytes 179.6 48.0 332.0 12.0 13.7(thawed) Primary human hepatocyte 299.2 104.2 314.4 50.4 13.2 after 4days in culture Primary Kupffer cells 3496.4 10713.5 83.7 2753.7 24.5(thawed)

Exposure of rat hepatocytes to TNFα and IFNγ in vitro promoted CXCL10mRNA and protein expression (Hassanshahi G, et al., Iran J AllergyAsthma Immunol 6(3):115-121 (2007)). Combinations of IL-1α/β, TNFα andIFNγ have been shown to induce CXCR3 ligand gene expression and proteinsecretion in intestinal cell lines and human intestinal xenografts. Toassess the epithelial contribution to the cytokine response, 5 ng/mLTNFα, 5 ng/mL IFNγ, or both, was added for presence of 24 hours tostimulate the gut epithelium (Caco2-BBE/HT29-MTX) basally. Co-treatmentof TNFα and IFNγ on the gut epithelium, in the absence of immune cells,resulted in marked amplification of 4 out of the 8 chemokines identifiedin the integrated system, including CXCL9, CXCL10, CXCL11 and CX3CL1(Table 11).

These results corroborated with the RNAseq findings and demonstratedthat IFNγ and TNFα signaling crosstalk was central to the chemokineproduction in the integrated system. These results showed epithelialcells are not passive bystanders during inflammatory gut-livercrosstalk, but contribute considerably to the overall immune milieu viaparacrine interactions with immune cells.

Under inflammatory gut-liver interaction, more than additiveamplification of chemokine production was detected from the disclosedintegrated gut-liver MPS. This amplification was in part mediated byTNFα and IFNγ signaling. Although immune cells were normally consideredas the primary sensor of endotoxin, the results here showed epithelialcells responded to immune cells-derived signals to influence CXCL9/10/11and CX3CL1 chemokine production. Exposure to TNFα and IFNγ did notresult in the amplification of CCL19, CCL20, CXCL6 and CCL2 inintestinal epithelial cells, which indicated the involvement ofadditional mechanisms, likely in different cell types.

The chemokine production observed in the integrated system can targetcells of the innate and adaptive immune system (Table 12). Potentialimmune cell recruitment can be inferred based on the chemokines and thecorresponding receptors profile. Although adaptive immunity was notrepresented in the system, regulation of pathways linking innate andadaptive immunity were evident during organ crosstalk. For example,enrichment of the CD40 costimulatory process was identified. CD40 is asurface receptor ubiquitously expressed on immune cells as well asnon-immune cells. CD40L is predominantly expressed by CD4⁺ T cells andCD40-CD40L engagement mediates heterologous cellular communication(Danese S, et al., Gut, 53(7):1035-1043 (2004)). Taken together, CXCR3chemokine production and CD40-CD40L regulation implicates a bias towardTh1 signaling.

Example 4. 4-Way MPS on the Chip for Pharmacokinetic/Pharmacodynamic(PK-PD) Prediction (1) 4-Way MPS Survival and Functional for at Least 2Week Materials & Methods

Validation: Flow rates in thirteen 4-MPS platforms (n=9 pumps perplatform) averaged from 0.82 to 1.12 μL/s without calibration, and hadan average standard deviation of 0.07 μL/s. Software calibration factorswere calculated from the flow rate measurement and entered to correctthe pump rates to within ±5% of the target flow rates.

A systemic interaction flow rate of Q_(mix)=5 mL/day was used for theduration of the experiment. Flow was partitioned to each MPS from themixer based on the relative percentages of cardiac output to each tissuetype in humans; these numbers can be easily modified on the platform fordifferent scaling strategies and MPS modules. Additionally, intra-MPSbasal recirculation rates of 0.25 μL/s (gut, lung, and endometrium MPSs)and 1 μL/s (liver MPS and mixer) were used to provide well-mixed basalmedia in each compartment and oxygenate the liver tissue. Complete mediachanges were conducted every 48 hours. During media changes, sampleswere taken from each compartment to assess MPS function throughout thetwo-week interaction study. Biomarker metrics of healthy cell functionwere measured during a 2-week co-culture of 4-way MPS: liver, gut, lung,and endometrium, with a partitioning of flow. Every two days, secretedalbumin and IGFBP-1 were measured from conditioned media. Barrierintegrity of the Gut and Lung MPSs was quantified with trans-epithelialelectrical resistance (TEER), measured off-platform using the commercialEndOhm systems. Simultaneously, functionality of each MPS in isolationwas monitored.

Results 1. 4-Way MPS Supports Cell Viability and Functions for at LeastTwo Weeks.

Continuous functionality metrics from 4-MPS platform studies indicatedthe multi-organ MPS viability during the 2-week culture. Transientalbumin secretion kinetics was observed of an initial increase inalbumin secretion followed by a gradual decline by the conclusion of theexperiment. Barrier integrity of the Gut and Lung MPSs was quantifiedwith trans-epithelial electrical resistance (TEER). TEER values from theGut MPS fluctuated in the early days of interaction studies beforesettling into a 150-250 Ω·cm² range for the remainder of the experiment.Lung MPS TEER values followed a similar trend of high TEER during thefirst few days, but eventually established stable values in the 600-800Ω·cm² range. Endometrium MPS functionality, evaluated by secretion ofinsulin-like growth factor-binding protein 1 (IGFBP-1), remained in the20-30 pg/day range throughout the study. Similar trends for eachphenotypic metric in the isolation studies were observed, but IGFBP-1secretion rate in the isolated endometrium MPS (off-platform) was lowerthan that of interaction studies.

2. Endogenously Produced Albumin from One Organ was UniformlyDistributed to Each Compartment with the Controlled Systemic Flow Rate.

In the 4-MPS platform, the effect of systemic flowrate (Q_(mix)) onalbumin (endogenously produced by liver MPS) secretion and distributionkinetics was characterized via collecting samples from each compartmentand, then, the results were computationally model to assess the accuracyof the distribution. The albumin concentrations in each compartment andthe mixing chamber were at day 2 (Q_(mix)=5 ml/day), day 4 (Q_(mix)=15ml/day), and day 6 (Q_(mix)=30 ml/day).

With an increasing systemic flow rate, albumin was distributed moreuniformly as demonstrated by experimental measurements, where thedeviation between MPSs was considerably lower with higher flow rates.Similarly, the calculated albumin secretion rates show smaller standarddeviations. However, one platform showed considerably lower albumin inall compartments for days 2-4. Furthermore, computationally generatedalbumin distribution profiles was compared with experimentally measuredalbumin concentrations. The ratios of both values indicated that thehigher flowrate resulted in more deterministic molecular biodistributionin the 4-way MPS platform.

(2) Gut-Liver/Lung/Endo 4-Way Platform: Independent Flow Rate ControlImproves PK-PD Prediction for Complex Physiology Background

In the study of modern medicine for human, interpretation of resultsfrom animal studies for the prospect of human treatment generallyemploys allometric scaling; and the interpretation of in vitro resultsfor the prospect of in vivo efficacy is commonly referred to IVIVCorrelation and Extrapolation. In vitro studies OF liver MPSpharmacokinetics (PK) is characterized by accounting for binding inmedia, drug uptake, and elimination. In vivo studies use known clinicaldata and physiological-based PK (PBPK)/absorption/binding models tocalculate comparable parameters.

Clinical PK data of seven drugs from Manvelian et al. 2012, Shimamoto etal. 2000, Yilmaz et al. 2011, Willis et al. 1979 were compared with invitro liver data gathered from LIVERCHIP™ to assess the predictionaccuracy of in vitro results from LIVERCHIP™. While PBPK in vivo forfree diclofenac elimination per cell has a rate constant of 1.76×10⁻¹⁰(cell*min)⁻¹, scaled liver MPS from LIVERCHIP™ was studied to show adiclofenac elimination rate constant of 5.66×10⁻⁹ (cell*min)⁻¹. Hence,in vitro drug PK data from LIVERCHIP™ overestimated in vivo drugelimination rate.

Materials & Methods

Following a diagram and flow partitioning (total Qmixing=1 μL/s;liver/mixer recirculation=1 μL/s; gut/lung/endometrium recirculationrate=0.5 μL/s) (9 pumped flows: 5 self-circ, 4 mixing;

6 independent flow rates: 5 self-circ flows collapsed to 2 independentpneumatic duty cycles; 6 pump sets=18 DOF), 4-way MPS interactome wasstudied, where addition of agents to the mixing chamber accounted for anintravenous dosage while addition to the gut chamber accounted for anoral dosage. Drug was added to the apical side of gut chamber for theexperiment.

Results

Uniform drug distribution was calculated as time for downstream(Endometrium) MPS to reach 90% of the concentration in mixing chamber.

Drug exposure was calculated as area under curve (AUC) from 0-48 hr indownstream (Endometrium) MPS. Drug exposure and distribution were ableto strongly drive selection of useful operational ranges: Qmixing>15mL/day for drug permeability greater than 10⁻⁶ cm/s, and Qmixing>40mL/day for AUC0-48 hr of greater than 2*10⁴ ng/L*hr.

Example 5. Operation of 7-Way MPS on the Chip Materials & Methods

Validation: Flow rates in ten 7-way platforms (n=17 pumps per platform)averaged 1.12±0.10 μL/s. Software calibration factors were calculatedfrom the flow rate measurement and entered to correct the pump rates towithin ±5% of the target flow rates (0.99±0.056 μL/s).

A 7-way MPS platform was utilized and operated in a similar manner tothe 4-MPS platform described in Example 4. The 7-way platform includegut (immune-competent), liver (immune-competent), lung, endometrium,cardiac, brain, and pancreas MPSs, and was assessed for survivabilityand function over a 3-week period. Each MPS was differentiated ormatured in isolation prior to the interaction study. Platforms were runat a systemic flow rate of Q_(mix)=10 mL/day, with flow partitioning.During the medium changes, a basal common medium was used for the gut,lung, liver, and endometrium MPSs, while the new MPS were supplied withtheir preferred maintenance media. Each basal medium was then allowed tomix throughout the course of the interaction, with media changes at48-hour intervals. Functionality of each MPS was evaluated every 2-4days up to 3 weeks, in comparison to isolated MPSs to benchmark thenon-interacting MPS functions. Due to the dramatic reduction in thefunctionality of isolated pancreas MPS, islets were replaced with thefresh islets at day 12 for both interaction and in-isolation studies.

Results 1. 7-Way MPS Supports Cell Viability and Functions for at LeastThree Weeks.

Transient albumin secretion kinetics, sustained gut and lung TEERvalues, and IGFBP-1 secretion profiles were established. Thefunctionality of cardiac MPS, which was monitored by beat frequency, waswell maintained during the study. N-acetyl aspartate (NAA) and c-peptiderelease profiles revealed that both the brain MPS and the pancreas werealso functional up to 3 weeks. The comparison of the interaction resultswith the isolation results showed no negative effect of interaction onthe MPS functionality. Increased NAA secretion and more sustainedc-peptide secretion were observed during the interaction. The long-termMPS viability and functionality could be maintained in the 7-MPSplatform for at least extended culture periods of three weeks.

2. “Orally” Administered Drug and its Metabolite were Distributed AcrossMPSs in Concentrations Consistent with Model PharmacokineticsPredictions.

Exogenous drug studies with clinically-relevant concentrations areimportant to translate in vitro results to clinical outcomes.Pharmacokinetics of diclofenac (DCF), a nonsteroidal anti-inflammatorydrug, was analyzed in the 7-MPS platform. The maximum measured plasmaconcentration, Cmax, of oral diclofenac in vivo varies between 2-6 μM(Davies N M, et al., Clin. Pharmacokinet. 33, 184-213 (1997)).4′-hydroxy-DCF (4-OH-DCF) is the common metabolite of DCF.

To recapitulate clinically observed Cmax from oral delivery in theplatform, diclofenac was added to the apical side of the gut MPS. Themeasured concentrations of DCF and 4′-hydroxy-DCF media across differentMPS compartments fitted respective pharmacokinetic model predictions.The DCF dose was absorbed across the gut epithelial barrier, distributedto the liver MPS and subsequently to the mixing chamber and all theother MPS compartments. Metabolite 4-OH-DCF was produced in the liverMPS, circulated across the 7-way MPS platform, and was detected in allthe others MPS compartments. Physiologically based pharmacokinetic(PBPK) model predictions on both DCF and 4-OH-DCF concentrations alignedwell with the measured data, which indicated the platform functions in adeterministic manner consistent with biology predictions. The unboundintrinsic clearance (CL_int(u); i.e., the ability of liver to removedrug in the absence of flow) was estimated to be 13.90 μL/min, andapproximately 19% of this clearance was estimated to be towards theformation of the 4-OH-DCF metabolite.

We claim:
 1. A fluidic multiwell device with an on-board pumping systemcomprising: a) a first plate comprising: two or more wells comprising athree-dimensional space in each well defined by a bottom surface and acircumferential wall; and an inlet and an outlet in each well; aspillway conduit positioned between the at least two wells, havingdefined geometries that allow unidirectional fluid connectivity fromabove the bottom surface of a first well to a second well; a network offluid paths providing fluid connectivity between at least two of thewells through the inlet and the outlet of each of the two wells; b) adetachable second plate comprising: a plurality of internal channels,each with an inlet opening and an outlet opening on opposing sides ofthe second plate, and one or more holes on the surface of the secondplate in connection with each of the internal channels; and c) a barriermembrane positioned between the fluid paths of the first plate and theone or more holes on the surface of the second plate, optionally bondedto the first plate, wherein the barrier membrane is at least partiallyflexible, such that applying a pressure to the internal channels of thesecond plate causes the membrane to move, thereby obstructing orclearing a portion of the fluid paths of the first plate.
 2. The deviceof claim 1 further comprising a pneumatic manifold.
 3. The device ofclaim 2 wherein the membrane is connected to the pneumatic manifold. 4.A self-leveling spillway in a multi-well cell culture system, thespillways comprising a geometry that causes capillary flow across thespillway for unidirectional self-level of fluid.
 5. The spillway ofclaim 4 comprising a step entry geometry.
 6. The spillway of claim 4comprising a V-cut to minimize fluid film disruption.
 7. The spillway ofclaim 4 comprising a radial meniscus pinning groove around the sourcewell.
 8. The spillway of claim 4 comprising a small-width and/or highaspect ratio groove at the bottom along the conduit effective to causespontaneous capillary flow.
 9. The spillway of claim 8 wherein theconduit comprises an enlarged curved area to break fluid film intodrops.
 10. The spillway of claim 9 wherein the conduit comprises avertical groove along the wall and toward the bottom of the destinationwell, optionally comprising an undercut into wall of the destinationwell positioned sufficiently from the exit of the conduit to preventback flow and syphoning.
 11. The spillway of claim 9 comprising ringsthat prevent adhesion of fluid.
 12. A fluidic multi-well device forculturing cells comprising an internal humidity sensor and/or a fluidmoat to maintain humidity.
 13. A fluidic multi-well device comprisingfluidic pumping channels and a central modular pump and manifold. 14.The device of claim 13 comprising a pump for pulsating flow.
 15. Thedevice of claim 13 comprising a pump for smooth flow in combination withparallel fluid channels.
 16. The device of claim 13 comprising pumpingmeans with a flow rate between zero and hundreds of milliliters per day,optionally with a controlled volume flux between 0.1 and 10microliter/stroke, and frequencies between about 0.1 Hz and 20 Hz. 17.The device of claim 13 comprising an oxygen or fluid level sensor oroptical means for determining fluid levels.
 18. The device of claim 13further comprising a fluid aggregation lid.
 19. The device of claim 12or 13 comprising a symmetrical front and back electrode capacitor foruse as a monitor.
 20. A perfusion-enabled removable scaffold for afluidic multi-well device.
 21. A system comprising fluidic multi-welldevices comprising any of the devices, spillways, or scaffolds of any ofclaims 1, 4, 12, 13, and 20, alone or in combination.
 22. The system ofclaim 21 further comprising organ or tissue specific cells in themulti-well devices.
 23. The system of claim 22 wherein the cells are ofa different origin in each of the multi-well devices.
 24. The system ofclaim 23 comprising a multi-well device with cells of different originin the same device.
 25. The system of claim 24 wherein the cells areselected from the group consisting of liver cells, intestinal cells,pancreatic cells, muscle cells, bladder cells, kidney cells, pluripotentcells, and hematopoietic cells.
 26. A method of culturing cellscomprising seeding the devices of any of claims 1, 4, 12, 13, and 20with cells.
 27. The method of claim 26 further comprising exposing thecells to an agent to determine its effect on the cells.
 28. The methodof claim 27 further comprising administering the agent in differentdosages, in a different dosing regimen, or in combination with one ormore other agents and determining its effect on the cells.
 29. Themethod of any of claim 28 wherein the agent is administered to differentcell types or cell types associated with one or more diseases ordisorders.